Production of functional antibodies in filamentous fungi

ABSTRACT

Described herein are methods for the production of monoclonal antibodies in filamentous fungi host cells. The monoclonal antibodies are expressed as full-length fusion proteins that retain functional antigen binding and antibody-dependent cellular cytotoxicity capabilities. Improvements in the cleavage of the glucoamylase-light chain fusion protein to yield a mature antibody are also provided. The antibodies produced in filamentous fungi show equivalent pharmacokinetic disposition to antibodies produced in mammalian cells.

RELATED APPLICATIONS

This is a divisional of application of Ser. No. 10/418,836, nowabandoned which claims priority to application No. 60/373,889, filedApr. 18, 2002; application No. 60/411,540, filed Sep. 18, 2002;application No. 60/411,537, filed Sep. 18, 2002, and application No.60/452,134, filed Mar. 4, 2003.

FIELD OF THE INVENTION

The present invention is directed to increased secretion ofimmunoglobulins from filamentous fungi. The invention discloses fusionnucleic acids, vectors, fusion polypeptides, and processes for obtainingthe immunoglobulins.

BACKGROUND OF THE INVENTION

Production of fusion polypeptides has been reported in a number oforganisms, including E. coli, yeast, and filamentous fungi. For example,bovine chymosin and porcine pancreatic prophospholipase A₂ have bothbeen produced in Aspergillus niger or Aspergillus niger var. awamori(previously known as Aspergillus awamon) as fusions to full-lengthglucoamylase (GAI) (U.S. Pat. No. 5,679,543; Ward et al., Bio/technology8:435-440, 1990; Roberts et al., Gene 122:155-161, 1992). Humaninterleukin 6 (hIL6) has been produced in A. nidulans as a fusion tofull-length A. niger GAI (Contreras et al., Biotechnology 9:378-381,1991). Hen egg white lysozyme (Jeenes et al., FEMS Microbiol. Lett.107:267-272, 1993) and human lactoferrin (Ward et al., Bio/technology13:498-503, 1995) have been produced in A. niger as fusions to residues1498 of glucoamylase and hIL6 has been produced in A. niger as a fusionto glucoamylase residues 1-514 (Broekhuijsen et al., J. Biotechnol.31:135-145, 1993). In some of the above experiments (Contreras et al.,1991; Broekhuijsen et al., 1993; Ward et al., 1995) a KEX2 proteaserecognition site (Lys, Arg) has been inserted between glucoamylase andthe desired polypeptide to allow in vivo release of the desiredpolypeptide from the fusion protein as a result of the action of anative Aspergillus KEX2-like protease (the Aspergillus KEX2-likeprotease is now designated KEXB).

Additionally, bovine chymosin has been produced in A. niger var. awamorias a fusion with full-length native alpha-amylase (Korman et al., Curr.Genet. 17:203-212, 1990) and in A. oryzae as a fusion with truncatedforms of A. oryzae glucoamylase (either residues 1-603 or 1-511;Tsuchiya et al., Biosci. Biotech. Biochem. 58:895-899, 1994).

A small protein (epidermal growth hormone; 53 amino acids) has beenproduced in Aspergillus as a tandem fusion of three copies of theprotein (U.S. Pat. No. 5,218,093). The trimer of EGF was secreted as aresult of the inclusion of an N-terminal secretion signal sequence.However, the EGF molecules were not additionally fused to a proteinefficiently secreted by filamentous fungi and no method for subsequentseparation of monomeric EGF proteins was provided.

The glaA gene encodes glucoamylase which is highly expressed in manystrains of Aspergillus niger and Aspergillus niger var. awamori. Thepromoter and secretion signal sequence of the gene have been used toexpress heterologous genes in Aspergilli including bovine chymosin inAspergillus nidulans and A. niger var. awamori as previously described(Cullen, D. et al., (1987) Bio/Technology 5, 713-719 and EPO PublicationNo. 0 215 594). In the latter experiments, a variety of constructs weremade, incorporating prochymosin cDNA, either the glucoamylase or thechymosin secretion signal and, in one case, the first 11 codons ofmature glucoamylase. Maximum yields of secreted chymosin obtained fromA. awamori were below 15 mg/l in 50 ml shake flask cultures and wereobtained using the chymosin signal sequence encoded by pGRG3. Theseprevious studies indicated that integrated plasmid copy number did notcorrelate with chymosin yields. Abundant polyadenylated chymosin mRNAwas produced, and intracellular levels of chymosin were high in sometransformants regardless of the source of secretion signal. It wasinferred that transcription was not a limiting factor in chymosinproduction but that secretion may have been inefficient. It was alsoevident that the addition of a small amino terminal segment (11 aminoacids) of glucoamylase to the propeptide of prochymosin did not preventactivation to mature chymosin. The amount of extracellular chymosinobtained with the first eleven codons of glucoamylase, however, wassubstantially less than that obtained when the glucoamylase signal wasused alone. Subsequently, it was demonstrated that chymosin productioncould be greatly increased when a fusion protein consisting offull-length glucoamylase and prochymosin was produced (U.S. Ser. No.08/318,494; Ward et al. Bio/technology 8:435-440, 1990).

Aspergillus niger and Aspergillus niger var. awamori (A. awamon)glucoamylases have identical amino acid sequences. The glucoamylase isinitially synthesized as preproglucoamylase. The pre and pro regions areremoved during the secretion process so that mature glucoamylase isreleased to the external medium. Two forms of mature glucoamylase arerecognized in culture supernatants: GAI is the full-length form (aminoacid residues 1-616) and GAII is a natural proteolytic fragmentcomprising amino acid residues 1-512. GAI is known to fold as twoseparate domains joined by an extended linker region. The two domainsare the 471 residue catalytic domain (amino acids 1-471) and the 108residue starch binding domain (amino acids 509-616), the linker regionbeing 36 residues in length (amino acids 472-508). GAII lacks the starchbinding domain. These details of glucoamylase structure are reviewed byLibby et al. (Protein Engineering 7:1109-1114, 1994) and are showndiagrammatically in FIG. 2.

Trichoderma reesei produces several cellulase enzymes, includingcellobiohydrolase I (CBHI), which are folded into two separate domains(catalytic and binding domains) separated by an extended linker region.Foreign polypeptides have been secreted in T. reesei as fusions with thecatalytic domain plus linker region of CBHI (Nyyssonen et al.,Bio/technology 11:591-595, 1993).

Antibody production has been, to date, preferably performed intransgenic animals, mammalian cell culture or plants. Each of thesemethods suffers from one or more drawbacks. For example, transgenicanimals and mammalian cell cultures each have a risk of beingcontaminated by viral or other adventitious agents, e.g., prions. Inaddition, the ability to scale up any one of these production systems islimited. Recombinant plants may take approximately ten months to producea recombinant protein, while mammalian cells may take about threemonths. Thus, there remains a need for alternative methods for antibodyproduction.

SUMMARY OF THE INVENTION

Provided herein are nucleic acids, cells and methods for the productionof immunoglobulins.

In a first embodiment, nucleic acids encoding a functional monoclonalimmunoglobulin are provided. In one aspect, a nucleic acid comprisingregulatory sequences operatively linked to a first, second, third andfourth nucleic acid sequences are provided. Terminator sequences areoptionally provided following the fourth nucleic acid sequence. In asecond aspect, the first nucleic acid sequence encodes a signalpolypeptide functional as a secretory sequence in a first filamentousfungus, the second nucleic acid encodes a secreted polypeptide orfunctional portion thereof normally secreted from said first or a secondfilamentous fungus, the third nucleic acid encodes a cleavable linkerand the fourth nucleic acid encodes an immunoglobulin chain or fragmentthereof.

In a third aspect, an expression cassette comprising nucleic acidsequences encoding an immunoglobulin chain is provided.

In a second embodiment, methods of expressing a functional monoclonalantibody are provided. In one aspect, a host cell is (i) transformedwith a first expression cassette comprising a nucleic acid sequenceencoding a first immunoglobulin chain, (ii) transformed with a secondexpression cassette comprising a nucleic acid sequence encoding a secondimmunoglobulin chain, and (iii) cultured under appropriate conditions toexpress the immunoglobulin chains. Optionally, the immunoglobulin chainsmay be recovered. In one aspect, the immunoglobulin chains are expressedas a fusion protein. The expressed fusion immunoglobulin chains aresubsequently assembled as functional antibodies and secreted.

In a third embodiment, cells capable of expressing an immunoglobulin areprovided. Host cells are transformed with two expression cassettes, afirst expression cassette encoding a first immunoglobulin chain type(e.g., either a heavy or light chain) and a second expression cassetteencoding a second immunoglobulin chain type (e.g., either light or heavychain, respectively). The heavy chain may be of any immunoglobulinclass.

In a fourth embodiment, a functional monoclonal immunoglobulin isprovided. In one aspect, the functional monoclonal antibody chains areexpressed as fusion proteins consisting of the glucoamylase signalsequence, prosequence, catalytic domain and linker region up to aminoacid number 502 of mature glucoamylase, followed by amino acids YKR andthen by the mature immunoglobulin chain. One chain may be either theheavy or light chain.

In a second aspect, the fully assembled antibodies are treated with aprotease to liberate an immunoglobulin from the fusion protein. In athird aspect, the antibodies may be treated with a deglycosylatingenzyme.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of an antibody. Indicated on thedrawing are the various regions of the antibody and the names of variousantibody fragments.

FIG. 2 is a diagram depicting the two forms of glucoamylase fromAspergillus niger or Aspergillus niger var. awamori.

FIG. 3 is a diagram of plasmid pQ83.

FIG. 4 is a diagram of plasmid pCL1.

FIG. 5 is a diagram of pCL5, a second trastuzumab heavy chain expressionplasmid.

FIG. 6 is a diagram of plasmid pCL2.

FIG. 7 is a diagram of plasmid pCL3.

FIG. 8 shows the results of SDS-PAGE under reducing conditions and withCoomassie Brilliant Blue staining of samples which had been purified byprotein A chromatography. The bands observed for transformant 1-LC/HC-3(lane 3) were identified as the light chain (25 kDa), non-glycosylatedand glycosylated forms of the heavy chain (50 and 53 kDa),glucoamylase-light chain fusion protein (85 kDa) and glucoamylase-heavychain fusion (116 kDa). The bands observed for transformant 1-HCA-4(lane 2) were identified as the light chain (25 kDa), non-glycosylatedform of the heavy chain (50 kDa), glucoamylase-light chain fusionprotein (85 kDa) and glucoamylase-heavy chain fusion (116 kDa). Thebands observed for transformant 1-Fab-1 (lane 1) were identified as thelight chain and Fd′ chain (both 25 kDa) and the glucoamylase-light chainand glucoamylase-Fd′ fusion proteins (both 85 kDa).

FIG. 9 shows the results of SDS-PAGE (NuPAGE Tris-AcetateElectrophoresis System from Invitrogen Corporation, Carlsbad, Calif.)under non-reducing conditions and with Coomassie Brilliant Blue stainingof samples which had been purified by protein A chromatography. Themajor bands observed for transformant 1-LC/HC-3 (lane 4) were identifiedas assembled IgG1 (150 kDa), assembled IgG1 with one molecule ofglucoamylase attached (˜200 kDa) and assembled IgG1 with two moleculesof glucoamylase attached (˜250 kDa). The major bands observed fortransformant 1-HCΔ-4 (lane 3) were identified as assembled IgG1 (150kDa), assembled IgG1 with one molecule of glucoamylase attached (˜200kDa) and assembled IgG1 with two molecules of glucoamylase attached(˜250 kDa). The major bands observed for transformant 1-Fab-1 (lane 2)were identified as assembled Fab′ (50 kDa) and assembled Fab′ with onemolecule of glucoamylase attached (˜100 kDa).

FIG. 10 shows the results of SDS-PAGE under reducing and non-reducingconditions of samples of Fab′ and F(ab′)2 purified from supernatant oftransformant 1-Fab-12 by hydrophobic charge induction chromatographyfollowed by size exclusion chromatography. A5, B11, B7 and B3 representdifferent fractions collected from the size exclusion chromatographycolumn.

FIG. 11 is a graph showing the anti-proliferative effect of the HER2antibodies on human breast adenocarcinoma cell line, SK-BR-3 (ATCCnumber: HTB-30). Commercial Herceptin antibodies are indicated bydiamonds (♦) and triangles (▴). Aspergillus transformant 1 LC/HC-3antibodies are indicated by circles (●) and squares (▪). Control cellswere A-431, a human epidermoid carcinoma that expresses high levels ofthe EGF receptor and low levels of HER2.

FIG. 12 is a graph showing the binding of Hu1D10 antibody derived fromNS0 mouse myeloma cell line (squares; ▪), and two Aspergillus producedantibodies (designated as An-3G-Hu1D10 [circles; ●] and An-Hu1D10[inverted triangles; ▾]) to Raji cells. No significant difference inbinding was observed.

FIG. 13 is a graph showing the competitive binding of FITC-labeledantibody with Hu1D10 antibody derived from NS0 mouse myeloma cell line(squares; ▪), and two Aspergillus produced antibodies (An-3G-Hu1D10[circles; ●] and An-Hu1D10 [inverted triangles; ▾]) to Raji cells. Nosignificant difference in binding was observed.

FIG. 14 is a bar graph indicating the percentage of cells in whichapoptosis has been induced by Hu1D10, An-3G-Hu1D10 and An-Hu1D10 at 5hours or 24 hours. No significant difference in inducing apoptosis wasobserved.

FIGS. 15 A and B are graphs depicting the levels of Antibody-DependentCellular Cytotoxicity reached by each of the three antibodies tested,i.e., Hu1D10, An-3G-Hu1D10 and An-Hu1D10, in two different donors. Clearindication of ADCC activity by Aspergillus-derived antibodies isexhibited.

FIG. 16 is a graph of the in vivo pharmacokinetics of CHO-derived andAspergillus-derived trastuzumab. No significant difference inpharmacokinetic disposition was observed for the fungal-derivedantibody.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that desired antibodies can be expressedand secreted in filamentous fungi at levels higher than that previouslyobtained using other expression systems.

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

DEFINITIONS

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid or polypeptide that is removed from at least onecomponent with which it is naturally associated.

An “expression cassette” or “expression vector” is a nucleic acidconstruct generated recombinantly or synthetically, with a series ofspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter. Expression cassette may be usedinterchangeably with DNA construct and its grammatical equivalents.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer nucleic acid sequences into cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes.

The term “nucleic acid molecule” or “nucleic acid sequence” includesRNA, DNA and cDNA molecules. It will be understood that, as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding a given protein may be produced.

As used herein, a “fusion DNA sequence” comprises from 5′ to 3′ first,second, third and fourth DNA sequences.

As used herein, “a first nucleic acid sequence” or “first DNA sequence”encodes a signal peptide functional as a secretory sequence in a firstfilamentous fungus. Such signal sequences include those fromglucoamylase, α-amylase and aspartyl proteases from Aspergillus nigervar. awamori, Aspergillus niger, Aspergillus oryzae, signal sequencesfrom cellobiohydrolase I, cellobiohydrolase II, endoglucanase I,endoglucanase III from Trichoderma, signal sequences from glucoamylasefrom Neurospora and Humicola as well as signal sequences from eukaryotesincluding the signal sequence from bovine chymosin, human tissueplasminogen activator, human interferon and synthetic consensuseukaryotic signal sequences such as that described by Gwynne et al.,(1987) Bio/Technology 5, 713-719. Particularly preferred signalsequences are those derived from polypeptides secreted by the expressionhost used to express and secrete the fusion polypeptide. For example,the signal sequence from glucoamylase from Aspergillus niger ispreferred when expressing and secreting a fusion polypeptide fromAspergillus niger. As used herein, first amino acid sequences correspondto secretory sequences which are functional in a filamentous fungus.Such amino acid sequences are encoded by first DNA sequences as defined.

As used herein, “second DNA sequences” encode “secreted polypeptides”normally expressed from filamentous fungi. Such secreted polypeptidesinclude glucoamylase, α-amylase and aspartyl proteases from Aspergillusniger var. awamori, Aspergillus niger, and Aspergillus oryzae,cellobiohydrolase I, cellobiohydrolase II, endoglucanase I andendoglucanase III from Trichoderma and glucoamylase from Neurosporaspecies and Humicola species. As with the first DNA sequences, preferredsecreted polypeptides are those which are naturally secreted by thefilamentous fungal expression host. Thus, for example when usingAspergillus niger, preferred secreted polypeptides are glucoamylase andα-amylase from Aspergillus niger, most preferably glucoamylase. In oneaspect the glucoamylase is greater than 95%, 96%, 97%, 98% or 99%homologous with an Aspergillus glucoamylase.

When Aspergillus glucoamylase is the secreted polypeptide encoded by thesecond DNA sequence, the whole protein or a portion thereof may be used,optionally including a prosequence. Thus, the cleavable linkerpolypeptide may be fused to glucoamylase at any amino acid residue fromposition 468-509. Other amino acid residues may be the fusion site bututilizing the above residues is particularly advantageous.

A “functional portion of a secreted polypeptide” or grammaticalequivalents means a truncated secreted polypeptide that retains itsability to fold into a normal, albeit truncated, configuration. Forexample, in the case of bovine chymosin production by A. niger var.awamori it has been shown that fusion of prochymosin following the 11thamino acid of mature glucoamylase provided no benefit compared toproduction of preprochymosin (U.S. Pat. No. 5,364,770). In U.S. Ser. No.08/318,494, it was shown that fusion of prochymosin onto the C-terminusof preproglucoamylase up to the 297th amino acid of mature glucoamylaseplus a repeat of amino acids 1-11 of mature glucoamylase yielded nosecreted chymosin in A. niger var. awamori. In the latter case it isunlikely that the portion (approximately 63%) of the glucoamylasecatalytic domain present in the fusion protein was able to foldcorrectly so that an aberrant, mis-folded and/or unstable fusion proteinmay have been produced which could not be secreted by the cell. Theinability of the partial catalytic domain to fold correctly may haveinterfered with the folding of the attached chymosin. Thus, it is likelythat sufficient residues of a domain of the naturally secretedpolypeptide must be present to allow it to fold in its normalconfiguration independently of the desired polypeptide to which it isattached.

In most cases, the portion of the secreted polypeptide will be bothcorrectly folded and result in increased secretion as compared to itsabsence.

Similarly, in most cases, the truncation of the secreted polypeptidemeans that the functional portion retains a biological function. In apreferred embodiment, the catalytic domain of a secreted polypeptide isused, although other functional domains may be used, for example, thesubstrate binding domains. In the case of Aspergillus niger andAspergillus niger var. awamori glucoamylase, preferred functionalportions retain the catalytic domain of the enzyme, and include aminoacids 1-471. Additionally preferred embodiments utilize the catalyticdomain and all or part of the linker region. Alternatively, the starchbinding domain of glucoamylase may be used, which comprises amino acids509-616 of Aspergillus niger and Aspergillus niger var. awamoriglucoamylase.

As used herein, “third DNA sequences” comprise DNA sequences encoding acleavable linker polypeptide. Such sequences include those which encodethe prosequence of bovine chymosin, the prosequence of subtilisin,prosequences of retroviral proteases including human immunodeficiencyvirus protease and DNA sequences encoding amino acid sequencesrecognized and cleaved by trypsin, factor X_(a) collagenase, clostripin,subtilisin, chymosin, yeast KEX2 protease, Aspergillus KEXB and thelike. See e.g. Marston, F. A. O. (1986) Biol. Chem J. 240, 1-12. Suchthird DNA sequences may also encode the amino acid methionine that maybe selectively cleaved by cyanogen bromide. It should be understood thatthe third DNA sequence need only encode that amino acid sequence whichis necessary to be recognized by a particular enzyme or chemical agentto bring about cleavage of the fusion polypeptide. Thus, the entireprosequence of, for example, chymosin or subtilisin need not be used.Rather, only that portion of the prosequence which is necessary forrecognition and cleavage by the appropriate enzyme is required.

It should be understood that the third nucleic acid need only encodethat amino acid sequence which is necessary to be recognized by aparticular enzyme or chemical agent to bring about cleavage of thefusion polypeptide.

Particularly preferred cleavable linkers are the KEX2 proteaserecognition site (Lys-Arg), which can be cleaved by a native AspergillusKEX2-like (KEXB) protease, trypsin protease recognition sites of Lys andArg, and the cleavage recognition site for endoproteinase-Lys-C.

As used herein, “fourth DNA sequences” encode “desired polypeptides.”Such desired polypeptides include mammalian immunoglobulin chains.Immunoglobulins include, but are not limited to, antibodies from anyspecies from which it is desirable to produce large quantities. It isespecially preferred that the antibodies are human antibodies.Immunoglobulins may be from any class, i.e., G, A, M, E or D. In anotheraspect the antibodies are monoclonal. The antibody chains may be eitherthe heavy or light chain. The terms “immunoglobulin” and “antibody” areused interchangeably herein.

The above-defined four DNA sequences encoding the corresponding fouramino acid sequences are combined to form a “fusion DNA sequence.” Suchfusion DNA sequences are assembled in proper reading frame from the 5′terminus to 3′ terminus in the order of first, second, third and fourthDNA sequences. As so assembled, the DNA sequence will encode a “fusionpolypeptide” or “fusion protein” encoding from its amino-terminus asignal peptide functional as a secretory sequence in a filamentousfungus, a secreted polypeptide or portion thereof normally secreted froma filamentous fungus, a cleavable linker polypeptide and a desiredpolypeptide.

As used herein, a “promotor sequence” is a DNA sequence which isrecognized by the particular filamentous fungus for expression purposes.It is operably linked to a DNA sequence encoding the above definedfusion polypeptide. Such linkage comprises positioning of the promoterwith respect to the translation initiation codon of the DNA sequenceencoding the fusion DNA sequence. The promoter sequence containstranscription and translation control sequences which mediate theexpression of the fusion DNA sequence. Examples include the promoterfrom the A. niger var. awamori or A. niger glucoamylase genes (Nunberg,J. H. et al. (1984) Mol. Cell. Biol. 4, 2306-2315; Boel, E. et al.(1984) EMBO J. 3, 1581-1585), the A. oryzae, A. niger var. awamori or A.niger or alpha-amylase genes, the Rhizomucor miehei carboxyl proteasegene, the Trichoderma reesei cellobiohydrolase I gene (Shoemaker, S. P.et al. (1984) European Patent Application No. EPO0137280A1), the A.nidulans trpC gene (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA81, 1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199,37-45) the A. nidulans alcA gene (Lockington, R. A. et al. (1986) Gene33 137-149), the A. nidulans amdS gene (McKnight, G. L. et al. (1986)Cell 46, 143-147), the A. nidulans amdS gene (Hynes, M. J. et al. (1983)Mol. Cell Biol. 3, 1430-1439), and higher eukaryotic promoters such asthe SV40 early promoter (Barclay, S. L. and E. Meller (1983) Molecularand Cellular Biology 3, 2117-2130).

Likewise a “terminator sequence” is a DNA sequence which is recognizedby the expression host to terminate transcription. It is operably linkedto the 3′ end of the fusion DNA encoding the fusion polypeptide to beexpressed. Examples include the terminator from the A. nidulans trpCgene (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474;Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45), the A. nigervar. awamori or A. niger glucoamylase genes (Nunberg, J. H. et al.(1984) Mol. Cell. Biol. 4, 2306-253; Boel, E. et al. (1984) EMBO J. 3,1581-1585), the A. oryzae, A. niger var. awamori or A. niger oralpha-amylase genes and the Rhizomucor miehei carboxyl protease gene(EPO Publication No. 0 215 594), although any fungal terminator islikely to be functional in the present invention.

A “polyadenylation sequence” is a DNA sequence which when transcribed isrecognized by the expression host to add polyadenosine residues totranscribed mRNA. It is operably linked to the 3′ end of the fusion DNAencoding the fusion polypeptide to be expressed. Examples includepolyadenylation sequences from the A. nidulans trpC gene (Yelton, M. etal. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474; Mullaney, E. J. etal. (1985) Mol. Gen. Genet. 199, 37-45), the A. niger var. awamori or A.niger glucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell. Biol.4, 2306-2315) (Boel, E. et al. (1984) EMBO J. 3, 1581-1585), the A.oryzae, A. niger var. awamori or A. niger or alpha-amylase genes and theRhizomucor miehei carboxyl protease gene described above. Any fungalpolyadenylation sequence, however, is likely to be functional in thepresent invention.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence which is capable of expressionin fungal cells and where expression of the selectable marker confers tocells containing the expressed gene the ability to grow in the presenceof a corresponding selective condition.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader is operably linked to DNA for a polypeptideif it is expressed as a preprotein that participates in the secretion ofthe polypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous, and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation. It follows thatthe term “Ig chain expression” refers to transcription and translationof the specific Ig chain gene to be expressed, the products of whichinclude precursor RNA, mRNA, polypeptide, post-translation processedpolypeptide, and derivatives thereof. Similarly, “Ig expression” refersto the transcription, translation and assembly of the Ig chains into aform exemplified by FIG. 1. By way of example, assays for immunoglobulinexpression include examination of fungal colonies when exposed to theappropriate conditions, western blot for Ig protein, as well as northernblot analysis and reverse transcriptase polymerase chain reaction(RT-PCR) assays for immunoglobulin mRNA.

As used herein the term “glycosylated” means that oligosaccharidemolecules have been added to particular amino acid residues on aprotein. A “de-glycosylated” protein is a protein that has been treatedto partially or completely remove the oligosaccharide molecules from theprotein. An “aglycosylated” protein is a protein that has not had theoligosaccharide molecules added to the protein. This may be due to amutation in the protein that prevents the addition of theoligosaccharide.

A “non-glycosylated” protein is a protein that does not have theoligosaccharide attached to the protein. This may be due to variousreasons, including but not limited to, the absence of enzymesresponsible for the addition of the oligosaccharides to proteins. Theterm “non-glycosylated” encompasses both proteins that have not had theoligosaccharide added to the protein and those in which theoligosaccharides have been added but were subsequently removed. An“aglycosylated” protein may be a “non-glycosylated” protein. A“non-glycosylated” protein may be either an “aglycosylated” protein or a“deglycosylated” protein.

Fusion Proteins

The above-defined four DNA sequences encoding the corresponding fouramino acid sequences are combined to form a “fusion DNA sequence.” Suchfusion DNA sequences are assembled in proper reading frame from the 5′terminus to 3′ terminus in the order of first, second, third and fourthDNA sequences. As so assembled, the DNA sequence will encode a “fusionpolypeptide” encoding from its amino-terminus a signal peptidefunctional as a secretory sequence in a filamentous fungus, a secretedpolypeptide or portion thereof normally secreted from a filamentousfungus, a cleavable linker polypeptide and a desired polypeptide, e.g.,an immunglobulin chain.

Antibodies are comprised of two chain types, one light and one heavy.The basic structure of an antibody is the same regardless of thespecificity for a particular antigen. Each antibody comprises fourpolypeptide chains of two different types. The chains are called theheavy chain (50-70 kDa in size) and the light chain (25 kDa). Twoidentical heavy chains and two identical light chains are linkedtogether via interchain disulphide bonds to create the antibody monomer(FIG. 1). In addition to the interchain disulphide bonds there are alsointrachain disulphide bonds in both the heavy and light chains.Different types of heavy and light chains are recognized. Heavy chainsmay be of the γ, ∝, α, δ or ε class and this defines the class ofimmunoglobulin, i.e., IgG, IgM, IgA, IgD or IgE respectively. There aresub-classes within these classes, e.g., in humans there are foursubclasses of the γ heavy chain, γ1, γ2, γ3 and γ4 to produce IgG1,IgG2, IgG3 and IgG4 respectively. Light chains may be of the λ or κ typebut this does not affect the class or subclass definition of theimmunoglobulin. Thus, a human IgG1 molecule will contain two identicalγ1 heavy chains linked to two identical light chains which may be of theλ or κ type (i.e., IgG1λ or IgG1κ).

A heavy chain is divided into distinct structural domains. For example,a γ heavy chain comprises, from the amino terminus, a variable region(VH), a constant region (CH1) a hinge region, a second constant region(CH2) and a third constant region (CH3). Light chains are structurallydivided into two domains, a variable region (VL) and a constant region(CL). Antibody forms in which the heavy chain has been truncated toremove some of the constant region can be generated by proteasedigestion or by recombinant DNA methodology. For example, Fab fragments(FIG. 1) of an IgG have a form of the heavy chain (Fd) lacking the hingeregion and the CH2 and CH3 domains whereas Fab′ fragments (FIG. 1) of anIgG have a form of the heavy chain (Fd′) which includes the hinge regionbut lacks the CH2 and CH3 domains.

Each chain will be expressed as a fusion protein by the host fungalcell. The chains are assembled into a complete antibody comprising thetwo heavy and two light chains.

Although cleavage of the fusion polypeptide to release the desiredantibody will often be useful, it is not necessary. Antibodies expressedand secreted as fusion proteins surprisingly assemble and retain theirantigen binding function.

Expression of Recombinant Immunoglobulin and Immunoglobulin Fragments

This invention provides filamentous fungal host cells which have beentransduced, transformed or transfected with an expression vectorcomprising a Ig-encoding nucleic acid sequence. The culture conditions,such as temperature, pH and the like, are those previously used for theparental host cell prior to transduction, transformation or transfectionand will be apparent to those skilled in the art.

In one approach, a filamentous fungal cell line is transfected with anexpression vector having a promoter or biologically active promoterfragment or one or more (e.g., a series) of enhancers which functions inthe host cell line, operably linked to a nucleic acid sequence encodingIg chain(s), such that the Ig chain(s) and fully assembled Ig isexpressed in the cell line. In a preferred embodiment, the DNA sequencesencode an Ig coding sequence. In another preferred embodiment, thepromoter is a regulatable one.

A. Nucleic Acid Constructs/Expression Vectors

Natural or synthetic polynucleotide fragments encoding immunoglobulin(“immunoglobulin-encoding nucleic acid sequences”) may be incorporatedinto heterologous nucleic acid constructs or vectors, capable ofintroduction into, and replication in, a filamentous fungal cell. Thevectors and methods disclosed herein are suitable for use in host cellsfor the expression of immunoglobulin chain(s) and fully assembledimmunoglobulin molecules. Any vector may be used as long as it isreplicable and viable in the cells into which it is introduced. Largenumbers of suitable vectors and promoters are known to those of skill inthe art, and are commercially available. Appropriate cloning andexpression vectors for use in filamentous fungal cells are alsodescribed in Sambrook et al., 1989, and Ausubel F M et al., 1989,expressly incorporated by reference herein. The appropriate DNA sequencemay be inserted into a plasmid or vector (collectively referred toherein as “vectors”) by a variety of procedures. In general, the DNAsequence is inserted into an appropriate restriction endonucleasesite(s) by standard procedures. Such procedures and related sub-cloningprocedures are deemed to be within the scope of knowledge of thoseskilled in the art.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as termination sequences. The vector may compriseregulatory sequences, including, for example, non-coding sequences, suchas introns and control elements, i.e., promoter and terminator elementsor 5′ and/or 3′ untranslated regions, effective for expression of thecoding sequence in host cells (and/or in a vector or host cellenvironment in which a modified soluble protein antigen coding sequenceis not normally expressed), operably linked to the coding sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, many of which are commercially available and/or aredescribed in Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and induciblepromoters, examples of which include a CMV promoter, an SV40 earlypromoter, an RSV promoter, an EF-1α promoter, a promoter containing thetet responsive element (TRE) in the tet-on or tet-off system asdescribed (ClonTech and BASF), the beta actin promoter and themetallothionein promoter that can upregulated by addition of certainmetal salts. In one embodiment of this invention, glaA promoter is used.This promoter is induced in the presence of maltose. Such promoters arewell known to those of skill in the art.

The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.Typical selectable marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, for example, ampicillin,methotrexate, tetracycline, neomycin (Southern and Berg, J., 1982),mycophenolic acid (Mulligan and Berg, 1980), puromycin, zeomycin, orhygromycin (Sugden et al., 1985) or (b) compliment an auxotrophicmutation or a naturally occuring nutritional deficiency in the hoststrain. In a preferred embodiment, a fungal pyrG gene is used as aselectable marker (Ballance, D. J. et al., 1983, Biochem. Biophys. Res.Commun. 112:284-289). In another preferred embodiment, a fungal amdSgene is used as a selectable marker (Tilburn, J. et al., 1983, Gene26:205-221).

A selected immunoglobulin coding sequence may be inserted into asuitable vector according to well-known recombinant techniques and usedto transform a cell line capable of immunoglobulin expression. Due tothe inherent degeneracy of the genetic code, other nucleic acidsequences which encode substantially the same or a functionallyequivalent amino acid sequence may be used to clone and express aspecific immunoglobulin, as further detailed above. Therefore it isappreciated that such substitutions in the coding region fall within thesequence variants covered by the present invention. Any and all of thesesequence variants can be utilized in the same way as described hereinfor a parent immunoglobulin-encoding nucleic acid sequence. One skilledin the art will recognize that differing immunoglobulins will be encodedby differing nucleic acid sequences.

Once the desired form of an immunoglobulin nucleic acid sequence,homologue, variant or fragment thereof, is obtained, it may be modifiedin a variety of ways. Where the sequence involves non-coding flankingregions, the flanking regions may be subjected to resection,mutagenesis, etc. Thus, transitions, transversions, deletions, andinsertions may be performed on the naturally occurring sequence.

Heterologous nucleic acid constructs may include the coding sequence foran immunoglobulin, or a variant, fragment or splice variant thereof: (i)in isolation; (ii) in combination with additional coding sequences; suchas fusion protein or signal peptide coding sequences, where theimmunoglobulin coding sequence is the dominant coding sequence; (iii) incombination with non-coding sequences, such as introns and controlelements, such as promoter and terminator elements or 5′ and/or 3′untranslated regions, effective for expression of the coding sequence ina suitable host; and/or (iv) in a vector or host environment in whichthe immunoglobulin coding sequence is a heterologous gene.

A heterologous nucleic acid containing the appropriate nucleic acidcoding sequence, as described above, together with appropriate promoterand control sequences, may be employed to transform filamentous fungalcells to permit the cells to express immunoglobulin chains and fullyassembled immunoglobulins.

In one aspect of the present invention, a heterologous nucleic acidconstruct is employed to transfer an immunoglobulin-encoding nucleicacid sequence into a cell in vitro, with established cell linespreferred. Preferably, cell lines that are to be used as productionhosts have the nucleic acid sequences of this invention stablyintegrated. It follows that any method effective to generate stabletransformants may be used in practicing the invention.

In one aspect of the present invention, the first and second expressioncassettes may be present on a single vector or on separate vectors.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,“Molecular Cloning: A Laboratory Manual”, Second Edition (Sambrook,Fritsch & Maniatis, 1989), “Animal Cell Culture” (R. I. Freshney, ed.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); and “Current Protocols in Immunology” (J. E. Coligan etal., eds., 1991). All patents, patent applications, articles andpublications mentioned herein both supra and infra, are hereby expresslyincorporated herein by reference.

B. Host Cells and Culture Conditions

The present invention provides cell lines comprising cells which havebeen modified, selected and cultured in a manner effective to result inexpression of immunoglobulin chain(s) and fully assembled immunoglobulinmolecules.

Examples of parental cell lines which may be treated and/or modified forimmunoglobulin expression include, but are not limited to, filamentousfungal cells. Examples of appropriate primary cell types for use inpracticing the invention include, but are not limited to, Aspergillusand Trichoderma.

Immunoglobulin expressing cells are cultured under conditions typicallyemployed to culture the parental cell line. Generally, cells arecultured in a standard medium containing physiological salts andnutrients, such as standard RPMI, MEM, IMEM or DMEM, typicallysupplemented with 5-10% serum, such as fetal bovine serum. Cultureconditions are also standard, e.g., cultures are incubated at 37° C. instationary or roller cultures until desired levels of immunoglobulinexpression are achieved.

Preferred culture conditions for a given cell line may be found in thescientific literature and/or from the source of the cell line such asthe American Type Culture Collection (ATCC). Typically, after cellgrowth has been established, the cells are exposed to conditionseffective to cause or inhibit the expression of immunoglobulin chain(s)and fully assembled immunoglobulin molecules.

In the preferred embodiments, where a immunoglobulin coding sequence isunder the control of an inducible promoter, the inducing agent, e.g., acarbohydrate, metal salt or antibiotics, is added to the medium at aconcentration effective to induce immunoglobulin expression.

C. Introduction of an Immunoglobulin-Encoding Nucleic Acid Sequence intoHost Cells

The invention further provides cells and cell compositions which havebeen genetically modified to comprise an exogenously providedimmunoglobulin-encoding nucleic acid sequence. A parental cell or cellline may be genetically modified (i.e., transduced, transformed ortransfected) with a cloning vector or an expression vector. The vectormay be, for example, in the form of a plasmid, a viral particle, aphage, etc, as further described above. In a preferred embodiment, aplasmid is used to transfect a filamentous fungal cell. Thetransformations may be sequential or by co-transformation.

Various methods may be employed for delivering an expression vector intocells in vitro. Methods of introducing nucleic acids into cells forexpression of heterologous nucleic acid sequences are also known to theordinarily skilled artisan, including, but not limited toelectroporation; nuclear microinjection or direct microinjection intosingle cells; bacterial protoplast fusion with intact cells; use ofpolycations, e.g., polybrene or polyornithine; membrane fusion withliposomes, lipofectamine or lipofection-mediated transfection; highvelocity bombardment with DNA-coated microprojectiles; incubation withcalcium phosphate-DNA precipitate; DEAE-Dextran mediated transfection;infection with modified viral nucleic acids; Agrobacterium-mediatedtransfer of DNA; and the like. In addition, heterologous nucleic acidconstructs comprising a immunoglobulin-encoding nucleic acid sequencecan be transcribed in vitro, and the resulting RNA introduced into thehost cell by well-known methods, e.g., by injection.

Following introduction of a heterologous nucleic acid constructcomprising the coding sequence for immunoglobulin chain(s), thegenetically modified cells can be cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants or amplifying expression of a immunoglobulin-encodingnucleic acid sequence. The culture conditions, such as temperature, pHand the like, are those previously used for the host cell selected forexpression, and will be apparent to those skilled in the art.

The progeny of cells into which such heterologous nucleic acidconstructs have been introduced are generally considered to comprise theimmunoglobulin-encoding nucleic acid sequence found in the heterologousnucleic acid construct.

Fungal Expression

Appropriate host cells include filamentous fungal cells. The“filamentous fungi” of the present invention, which serve both as theexpression hosts and the source of the first and second nucleic acids,are eukaryotic microorganisms and include all filamentous forms of thesubdivision Eumycotina, Alexopoulos, C. J. (1962), IntroductoryMycology, New York: Wiley. These fungi are characterized by a vegetativemycelium with a cell wall composed of chitin, glucans, and other complexpolysaccharides. The filamentous fungi of the present invention aremorphologically, physiologically, and genetically distinct from yeasts.Vegetative growth by filamentous fungi is by hyphal elongation. Incontrast, vegetative growth by yeasts such as S. cerevisiae is bybudding of a unicellular thallus. Illustrations of differences betweenS. cerevisiae and filamentous fungi include the inability of S.cerevisiae to process Aspergillus and Trichoderma introns and theinability to recognize many transcriptional regulators of filamentousfungi (Innis, M. A. et al. (1985) Science, 228, 21-26).

Various species of filamentous fungi may be used as expression hostsincluding the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Phanerochaete, Podospora, Endothia,Mucor, Fusarium, Humicola, and Chrysosporium. Specific expression hostsinclude A. nidulans, (Yelton, M., et al. (1984) Proc. Natl. Acad. Sci.USA, 81, 1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199,3745; John, M. A. and J. F. Peberdy (1984) Enzyme Microb. Technol. 6,386-389; Tilburn, et al. (1982) Gene 26, 205-221; Ballance, D. J. etal., (1983) Biochem. Biophys. Res. Comm. 112, 284-289; Johnston, I. L.et al. (1985) EMBO J. 4, 1307-1311) A. niger, (Kelly, J. M. and M. Hynes(1985) EMBO 4, 475-479) A. niger var. awamori, e.g., NRRL 3112, ATCC22342, ATCC 44733, ATCC 14331 and strain UVK 143f, A. oryzae, e.g., ATCC11490, N. crassa (Case, M. E. et al. (1979) Proc. Natl. Acad. Scie. USA76, 5259-5263; Lambowitz U.S. Pat. No. 4,486,553; Kinsey, J. A. and J.A. Rambosek (1984) Molecular and Cellular Biology 4, 117-122; Bull, J.H. and J. C. Wooton (1984) Nature 310, 701-704), Trichoderma reesei,e.g. NRRL 15709, ATCC 13631, 56764, 56765, 56466, 56767, and Trichodermaviride, e.g., ATCC 32098 and 32086. A preferred expression host is A.niger var. awamori in which the gene encoding the major secretedaspartyl protease has been deleted. The production of this preferredexpression host is described in U.S. patent application Ser. No. 214,237filed Jul. 1, 1988, expressly incorporated herein by reference.

During the secretion process in fungi, which are eukaryotes, thesecreted protein crosses the membrane from the cytoplasm into the lumenof the endoplasmic reticulum (ER). It is here that the protein folds anddisulphide bonds are formed. Chaperone proteins such as BiP and proteinslike protein disulphide isomerase assist in this process. It is also atthis stage where sugar chains are attached to the protein to produce aglycosylated protein. Sugars are typically added to asparagine residuesas N-linked glycosylation or to serine or threonine residues as O-linkedglycosylation. Antibodies are known to assemble in the ER. In mammaliancells the heavy chains become associated with BiP immediately on entryinto the ER and are not released until they have associated with thelight chain. Correctly folded and glycosylated proteins pass from the ERto the Golgi apparatus where the sugar chains are modified and where theKEX2 or KEXB protease of yeast and fungi resides. The N-linkedglycosylation added to secreted proteins produced in fungi differs fromthat added by mammalian cells.

Antibodies produced by the filamentous fungal host cells may be eitherglycosylated or non-glycosylated (i.e., aglycosylated ordeglycosylated). Because the fungal glycosylation pattern differs fromthat produced by mammalian cells, the antibodies may be treated with anenzyme to deglycosylate the antibody. Enzymes useful for suchdeglycosylation are endoglycosidase H, endoglycosidase F1,endoglycosidase F2, endoglycosidase A, PNGase F, PNGase A, and PNGaseAt.

We have surprisingly found that high levels of full-length assembledantibody can be made in fungi when both the heavy and light chains arefused to a native secreted protein. From the information provided aboveit is clear that the antibody would be expected to assemble in the ERwhen glucoamylase was still attached to the N-termini of each of thefour chains. This would produce a very large and complicated assembledprotein of greater than 350 kD. The glucoamylase would not be expectedto be cleaved from the antibody until the assembled complex passedthrough the Golgi apparatus.

Using the present inventive methods and host cells, we have attainedsurprisingly high levels of expression. The vast majority of reports ofantibody production in microbial systems, e.g., Escherichia coli oryeasts such as Saccharomyces cerevisiae or Pichia pastoris have involvedthe production of antibody fragments (e.g., Fab fragments) orsingle-chain antibody forms (e.g., ScFv) (Verma, R. et al., 1998, J.Immunological Methods 216:165-181; Pennell, C. A. and Eldin, P., 1998,Res. Immunol. 149:599-603;). A low level of full-length antibody hasbeen produced and secreted in Saccharomyces cerevisiae. In one study,100 ng/ml of light chain and 50-80 ng/ml of heavy chain were detected inthe culture supernatant and approximately 50-70% of the heavy chainswere associated with light chain (Horwitz, A. H. et al., 1988, Proc.Natl. Acad. Sci USA). Full-length antibody in a correctly assembled formhas been produced in the yeast Pichia pastoris (WO 00/23579). However,the highest yields reported were 36 mg/l.

In contrast, the system utilized herein has achieved levels ofexpression and secretion of greater than 0.5 g/l of full-lengthantibody. It is routinely found that greater than 1 g/l of the antibodymay be recovered from the fermentation broth. Reproducible levels of 1.5g/l have been achieved. Expression and/or secretion levels as high as 2to 3 g/l of full length antibody may be attained once the optimalconditions are in place. Although the antibody is secreted as a fusionprotein the antibody levels given herein have been corrected forglucoamylase. Thus, the absolute protein produced comprising an antibodyis greater than those stated; the amount produced has had thecontribution of glucoamylase subtracted to give the stated amounts.

Utility

For some applications of immunoglobulins it is of high important thatthe immunoglobulins are extremely pure, e.g. having a purity of morethan 99%. This is particularly true whenever the immunoglobulin is to beused as a therapeutic, but is also necessary for other applications.

Therapeutic and prophylactic vaccine compositions are contemplated,which generally comprise mixtures of one or more of the above-describedmonoclonal antibodies, including fragments thereof and combinationsthereof. Passive immunization by intramuscularly injection ofimmunoglobulin concentrates is a well-known application for temporaryprotection against infectious diseases, which is typically applied whenpeople are traveling from one part of the world to the other.

A more sophisticated application of antibodies for therapeutic use isbased on so called “drug-targeting” where very potent drugs arecovalently linked to antibodies with specific binding affinities towardsspecific cells in the human organism, e.g. cancer cells.

The above-described recombinant monoclonal antibodies, including Fabmolecules, Fv fragments as well as Fab′ and F(ab′)₂, which are capableof reacting immunologically with samples containing antigen particlesare also used herein to detect the presence of antigens in specificbinding assays of biological samples. In particular, the novelmonoclonal antibodies of the present invention can be used in highlysensitive methods of screening for the presence of an antigen.

The format of specific binding assays will be subject to a great deal ofvariation in accordance with procedures that are well known in the art.For example, specific binding assays can be formatted to utilize one, ora mixture of several, of the recombinant monoclonal antibodies,(including Fab molecules, Fv fragments as well as Fab′ and F(ab′)₂) thathave been prepared according to the present invention. The assay formatcan be generally based, for example, upon competition, direct bindingreaction or sandwich-type assay techniques. Furthermore, the presentassays can be conducted using immunoprecipitation or other techniques toseparate assay reagents during, or after commencement of, the assay.Other assays can be conducted using monoclonal antibodies that have beeninsolubilized prior to commencement of the assay. In this regard, anumber of insolubilization techniques are well known in the art,including, without limitation, insolubilization by adsorption to animmunosorbent or the like, absorption by contact with the wall of areaction vessel, covalent crosslinking to insoluble matrices or “solidphase” substrates, noncovalent attachment to solid phase substratesusing ionic or hydrophobic interactions, or by aggregation usingprecipitants such as polyethylene glycol or cross-linking agents such asglutaraldehyde.

There are a large number of solid phase substrates which can be selectedfor use in the present assays by those skilled in the art. For example,latex particles, microparticles, magnetic-, para-magnetic- ornonmagnetic-beads, membranes, plastic tubes, walls of microtitre wells,glass or silicon particles and sheep red blood cells all are suitablefor use herein.

In general, most of the present assays involve the use of a labeledbinding complex formed from the combination of a monoclonal antibody(including fragments thereof) with a detectable label moiety. A numberof such labels are known in the art and can be readily attached (eitherusing covalent or non-covalent association techniques) to the monoclonalantibodies of the present invention to provide a binding complex for usein the above-noted assay formats. Suitable detectable moieties include,but are not limited to, radioactive isotopes, fluorescers, luminescentcompounds (e.g., fluorescein and rhodamine), chemiluminescers (e.g.,acridinium, phenanthridinium and dioxetane compounds), enzymes (e.g.,alkaline phosphatase, horseradish peroxidase and beta-galactosidase),enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, and metalions. These labels can be associated with the antibodies usingattachment techniques that are known in the art.

Exemplary assay methods generally involve the steps of: (1) preparingthe detectably labeled binding complexes as above; (2) obtaining asample suspected of containing antigen; (3) incubating the sample withthe labeled complexes under conditions which allow for the formation ofan antibody-antigen complex; and (4) detecting the presence or absenceof labeled antibody-antigen complexes. As will be appreciated by thoseskilled in the art upon the reading of this specification, such assayscan be used to screen for the presence of antigens in human donor bloodand serum products. When the assays are used in the clinical settingsamples can be obtained from human and animal body fluids, such as wholeblood, serum, plasma, cerebrospinal fluid, urine and the like.Furthermore, the assays can be readily used to provide quantitativeinformation using reference to standards or calibrants as known in theart.

In one particular assay method of the invention, an enzyme-linkedimmunosorbent assay (ELISA) can be used to quantify an antigenconcentration in a sample. In the method, the specific binding moleculesof the present invention are conjugated to an enzyme to provide alabeled binding complex, wherein the assay uses the bound enzyme as aquantitative label. In order to measure antigen, a binding moleculecapable of specifically binding the selected antigen (e.g., an antibodymolecule) is immobilized to a solid phase substrate (e.g., a microtitreplate or plastic cup), incubated with test sample dilutions, washed andincubated with the binding molecule-enzyme complexes of the invention,and then washed again. In this regard, suitable enzyme labels aregenerally known, including, for example, horseradish peroxidase. Enzymeactivity bound to the solid phase is measured by adding the specificenzyme substrate, and determining product formation or substrateutilization calorimetrically. The enzyme activity bound to the solidphase substrate is a direct function of the amount of antigen present inthe sample.

In another particular assay method of the invention, the presence ofantigen in a biological sample (e.g., as an indicator of infection) canbe detected using strip immunoblot assay (SIA) techniques, such as thoseknown in the art which combine traditional Western and dot blottingtechniques, e.g., the RIBA™ (Chiron Corp., Emeryville, Calif.) test. Inthese assays, one or more of the specific binding molecules (therecombinant monoclonal antibodies, including Fab molecules) areimmobilized as individual, discrete bands on a membranous support teststrip. Visualization of reactivity with antigens present in thebiological sample is accomplished using sandwich binding techniques withlabeled antibody-conjugates in conjunction with a colorimetric enzymesubstrate. Internal controls can also be present on the strip. The assaycan be performed manually or used in an automated format.

Furthermore, the recombinant human monoclonal antibodies, (including Fabmolecules, Fv fragments as well as Fab′ and F(ab′)₂ molecules) that havebeen prepared according to the present invention can be used in affinitychromatography techniques in order to detect the presence of antigen ina biological sample or to purify the antigen from the other componentsof the biological sample. Such methods are well known in the art.

Kits suitable for use in conducting any of the above-described assaysand affinity chromatography techniques, and containing appropriatelabeled binding molecule complex reagents can also be provided inaccordance with the practice of the invention. Assay kits are assembledby packaging the appropriate materials, including all reagents andmaterials necessary for conducting the assay in a suitable container,along with an appropriate set of assay instructions.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and practice the presentinvention. They should not be considered as limiting the scope and/orspirit of the invention, but merely as being illustrative andrepresentative thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds); Ci (Curies) mCi (milliCuries); μCi (microCuries); TLC(thin layer achromatography); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms(mesyl); Et (ethyl), Me (methyl), SDS (sodium dodecyl sulfate), PAGE(polyacrylamide gel electrophoresis), kDa (kiloDaltons), bp (basepairs).

Example 1 Cloning DNA Encoding the Human Ig κ Light Chain ConstantRegion

The human Ig K light chain constant region was PCR amplified from humanleukocyte cDNA (QUICK-Clone cDNA, Clontech Laboratories, Palo Alto,Calif.). The primers used were:

BPF001: 5-CCGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTG-3 (SEQ ID NO:  1)BPF002: 5-CAGTTCTAGAGGATCAACACTCTCCCCTGTTGAAGCTCTTTG-3 (SEQ ID NO:  2)

BPF001 includes two silent mutations to introduce a NarI restrictionsite (GGCGCC) for cloning purposes. BPF002 introduces an XbaIrestriction site (TCTAGA) following the translation termination signalfor cloning purposes. The PCR product was cloned into pCR2.1 TOPO(Invitrogen Corporation, Carlsbad, Calif.) using the TOPO TA cloning kitand protocol supplied by the manufacturer to create K1-pCR2.1TOPO. DNAfrom clone K1-pCR2.1TOPO was sequenced. The sequence is shown below.

GAATTCGCCCTTCCGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAG (SEQ ID NO:  3)CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATCCTCTAGAACTGAAGGGCGAATTC

The sequence obtained matches GenBank accession number J00241; human Iggermline κ L chain, C region (inv3 allele).

Example 2 Cloning DNA Encoding the Human γ1 Heavy Chain Constant Region

The human γ 1 heavy chain constant region was PCR amplified from humanleukocyte cDNA (QUICK-Clone cDNA, Clontech Laboratories, Palo Alto,Calif.). The primers used were:

BPF006: 5-GGGCCCATCGGTCTTCCCCCTGGCA-3 (SEQ ID NO:  4) and BPF004:5-CAGTTCTAGAGGATCATTTACCCGGAGACAGGGAGAGGCTC-3 (SEQ ID NO:  5)

BPF006 takes advantage of the naturally occurring ApaI restriction site(GGGCCC) at the 5 end of the human γ1 CH1 region. BPF004 introduces anXbaI restriction site (TCTAGA) following the translation terminationcodon for cloning purposes. The PCR product was cloned into pCR2.1 TOPO(Invitrogen Corporation, Carlsbad, Calif.) using the TOPO TA cloning kitand protocol supplied by the manufacturer to create BG13-pCR2.1-TOPO.DNA from clone BG13-pCR2.1-TOPO was sequenced. The sequence is shownbelow.

GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA (SEQ ID NO:  6)GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATCCTCTAGA

The sequence obtained matches the exon sequences of GenBank accessionnumber Z17370; human germline immunoglobulin γ1 chain constant regiongene, except for the following:

-   -   1) A change from an A to a G at nucleotide number 500 in Z17370        which represents a Lysine to an Arginine change in the protein        corresponding to the G1m(3) allotype.    -   2) Changes from a T to a G and from a C to an A at nucleotide        numbers 1533 and 1537 respectively in Z17370 representing        Aspartate to Glutamate and Leucine to Methionine changes        respectively. These changes correspond to the non(1) allotype.    -   3) a silent mutation of C to T corresponding to base 1686 of        Z17370.

Example 3 Synthesis of DNA Encoding Trastuzumab Light Chain VariableRegion

DNA encoding the amino acid sequence of the trastuzumab light chainvariable region (As given in Carter et al., 1992, Proc. Natl. Acad. Sci.USA 89:4285-4289 except that tyrosine replaced glutamic acid at aminoacid position 55) was synthesized by Aptagen, Inc., Herndon, Va., usingtheir Gene Forge custom gene synthesis technology. The sequence is shownbelow.

TACGTATAAGCGCGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTG (SEQ ID NO:  7)TGGGCGATAGGGTCACTATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCCGGAAAGGCCCCGAAACTGCTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTTCCCGCTCTGGGACGGATTTCACTCTGACCATCAGCTCCCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACACTATACTACTCCTCCGACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTA CCGTGGCGGCGCC

This DNA sequence includes a 5′ SnaB1 restriction site (TACGTA) to allowdigestion and ligation to the A. niger glucoamylase coding regionfollowed by codons for the amino acids Lysine and Arginine (AAG CGC)representing a KEX2 protease cleavage site. At the 3′ end there is aNarI restriction site to allow digestion and ligation to the light chainconstant region. The codon usage in this DNA reflects the frequency ofcodon usage observed in Aspergillus genes.

Example 4 Synthesis of DNA Encoding Trastuzumab Heavy Chain VariableRegion

DNA encoding the amino acid sequence of the trastuzumab heavy chainvariable region (As given in Carter et al., 1992, Proc. Natl. Acad. Sci.USA 89:4285-4289 except that tyrosine replaced valine at amino acidposition 105) was synthesized by Aptagen, Inc., Herndon, Va., usingtheir Gene Forge custom gene synthesis technology. The sequence is shownbelow.

TACGTATAAGCGCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCC (SEQ ID NO:  8)GGGGGCTCTCTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATCCACTGGGTGCGTCAGGCTCCGGGTAAGGGCCTGGAGTGGGTTGCAAGGATTTATCCTACGAATGGTTATACTCGTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACTTCGAAAAACACAGCCTACCTCCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTAGCAGATGGGGTGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGTACACTAGTCACCGTCAGCAGCGCTAGCACCAAGGGCCC

This DNA sequence includes a 5′ SnaB1 restriction site (TACGTA) to allowdigestion and ligation to the A. niger glucoamylase coding regionfollowed by codons for the amino acids Lysine and Arginine (AAG CGC)representing a KEX2 protease cleavage site. At the 3′ end there is anApaI restriction site to allow digestion and ligation to the heavy chainconstant region. The codon usage in this DNA reflects the frequency ofcodon usage observed in Aspergillus genes.

Example 5 Construction of a Trastuzumab Light Chain Expression PlasmidContaining the pyrG Marker

The expression plasmid used for light chain expression in Aspergilluswas based on pGAMpR, a glucoamylase-prochymosin expression vector whichis described in detail in U.S. Pat. No. 5,679,543. This plasmid wasdigested with the restriction endonucleases SnaBI and XbaI, each ofwhich cuts only once in pGAMpR. SnaBI cuts the plasmid within the codingregion for the glucoamylase linker region and XbaI cuts pGAMpR justafter the 3 end of the chymosin coding region. Using techniques known inthe art the DNA sequences encoding the light chain variable and constantregions were assembled and inserted into pGAMpR replacing the chymosinencoding region. The final plasmid was named pQ83 (FIG. 3). This plasmidcontains the Neurospora crassa pyr4 gene as a selectable marker fortransformation into Aspergillus or other fungi. The Aspergillus awamoriglaA (glucoamylase) promoter and A. niger glaA terminator are includedto control expression of the open reading frame which includes the lightchain encoding DNA. This plasmid was designed for the expression of afusion protein consisting of the glucoamylase signal sequence,prosequence, catalytic domain and linker region up to amino acid number502 of mature glucoamylase (Nunberg, J. H. et al., 1984, Mol. Cell.Biol. 4:2306-2315), followed by amino acids YKR and then by the maturelight chain. Free light chain can be obtained after cleavage of thisfusion protein immediately after the KR residues placed at the end ofthe glucoamylase linker region by Aspergillus KEX2 proteinase. Thecomplete amino acid sequence of the fusion protein is given here. TheYKR sequence between the end of the glucoamylase linker region and thestart of the light chain sequence is underlined.

MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSGADSGI(SEQ ID NO:  9)VVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKTSTYKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 6 Construction of Trastuzumab Heavy Chain Expression Plasmids

The Aspergillus expression vector pGAMpR was modified by methods knownin the art to replace the N. crassa pyr4 gene with the Aspergillusnidulans amdS gene as a selectable marker for transformation intoAspergillus or other fungi (Kelly J. M. and Hynes, M. J., 1985, EMBO J.4:475-479; Corrick, C. M. et al., 1987, Gene 53:63-71). Using techniquesknown in the art the DNA sequences encoding the heavy chain variable andconstant regions were assembled and inserted into the version of theAspergillus expression vector pGAMpR with the amdS selectable marker.The final plasmid was named pCL1 (FIG. 4). The Aspergillus awamori glaA(glucoamylase) promoter and A. niger glaA terminator are included tocontrol expression of the open reading frame which includes the heavychain encoding DNA. This plasmid was designed for the expression of afusion protein consisting of the glucoamylase signal sequence,prosequence, catalytic domain and linker region up to amino acid number502 of mature glucoamylase, followed by amino acids YKR and then by themature heavy chain. Free heavy chain can be obtained after cleavage ofthis fusion protein immediately after the KR residues placed at the endof the glucoamylase linker region by Aspergillus KEX2 proteinase. Thecomplete amino acid sequence of the fusion protein is given here. TheYKR sequence between the end of the glucoamylase linker region and thestart of the heavy chain sequence is underlined.

MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSGADSGI(SEQ ID NO: 10)VVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKTSTYKREVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

A second trastuzumab heavy chain expression plasmid (pCL5; FIG. 5) wasconstructed which contained exactly the same expression cassette as pCL1(i.e., the Aspergillus niger var. awamori glaA promoter and A. nigerglaA terminator controlling expression of the open reading frameencoding a fusion protein consisting of the glucoamylase signalsequence, prosequence, catalytic domain and linker region up to aminoacid number 502 of mature glucoamylase, followed by amino acids YKR andthen by the mature heavy chain). The only differences between pCL1 andpCL5 were that the latter plasmid lacked the A. nidulans amdS gene and,therefore, lacked a fungal transformation marker and that pBR322 insteadof pUC100 was used as the bacterial plasmid backbone.

Example 7 Construction of an Expression Plasmid for the Fd′ Fragment ofthe Trastuzumab Heavy Chain

PCR was used to generate a DNA fragment encoding the Fd′ portion of theheavy chain (heavy chain truncated after the antibody hinge region)using the assembled heavy chain variable and constant region DNA astemplate. The following two primers were used: oligo1 (5′-AAC AGC TATGAC CAT G-3′) (SEQ ID NO: 11) and oligo2 (5-TCT AGA GGA TCA TGC GGC GCACGG TGG GCA TGT GTG AG-3) (SEQ ID NO: 12). The amplified 900 bp fragmentwas purified, digested with SnaBI and XbaI and the 719 bp SnaB1 to XbaIfragment generated was cloned into version of the the Aspergillusexpression pGAMpR with the amdS gene as selectable marker. The finalplasmid was named pCL2 (FIG. 6). The Aspergillus awamori glaA(glucoamylase) promoter and A. niger glaA terminator are included tocontrol expression of the open reading frame which includes the heavychain encoding DNA. This plasmid was designed for the expression of afusion protein consisting of the glucoamylase signal sequence,prosequence, catalytic domain and linker region up to amino acid number502 of mature glucoamylase, followed by amino acids YKR and then by themature Fd′ portion of the heavy chain. Free Fd′ chain can be obtainedafter cleavage of this fusion protein immediately after the KR residuesplaced at the end of the glucoamylase linker region by Aspergillus KEX2proteinase. The complete amino acid sequence of the fusion protein isgiven here. The YKR sequence between the end of the glucoamylase linkerregion and the start of the heavy chain sequence is underlined.

MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSGADSGI(SEQ ID NO: 13)VVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKTSTYKREVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCAA

Example 8 Construction of an Expression Plasmid for an AglycosylatedForm of the Trastuzumab Heavy Chain

There is known to be a single asparagine (at position 297) in the IgGheavy chain constant region which is the site of attachment for N-linkedglycosylation. In order to prevent glycosylation of the antibodyproduced in Aspergillus the codon encoding this asparagine has beenchanged to a codon which encodes glutamine. The QuikChange Site-DirectedMutagenesis kit (Stratagene, La Jolla, Calif.) was used according to themanufacturers directions to make the appropriate change to the DNAsequence. A plasmid containing the assembled DNA encoding the heavychain variable and constant regions was used as a template. Thefollowing two primers, one complementary to one DNA strand of theplasmid and the other complementary to the second strand of the plasmid,which overlap the asparagine codon to be mutated were used in themutagenesis procedure: 5-GAG CAG TAC CAG AGC ACG TAC CGT GTG GTC-3 (SEQID NO: 14) and 5-GTA CGT GCT CTG GTA CTG CTC CTC CCG CGG CT-3 (SEQ IDNO: 15). The altered codon is underlined. DNA sequence analysisconfirmed that the desired sequence change had been created and that noother undesired mutations had been introduced. The mutated version offull-length heavy chain was then cloned into the version of theAspergillus expression vector pGAMpR with the amdS selectable marker.The final plasmid was named pCL3 (FIG. 7). The Aspergillus awamori glaA(glucoamylase) promoter and A. niger glaA terminator are included tocontrol expression of the open reading frame which includes the heavychain encoding DNA. This plasmid was designed for the expression of afusion protein consisting of the glucoamylase signal sequence,prosequence, catalytic domain and linker region up to amino acid number502 of mature glucoamylase (Nunberg et al., 1984, Mol. Cell. Biol.4:2306-2315), followed by amino acids YKR and then by the mature heavychain containing the mutation to prevent glycosylation. Free heavy chaincan be obtained after cleavage of this fusion protein immediately afterthe KR residues placed at the end of the glucoamylase linker region byAspergillus KEX2 proteinase. The complete amino acid sequence of thefusion protein is given here. The YKR sequence between the end of theglucoamylase linker region and the start of the heavy chain sequence isunderlined as is the glutamine residue which replaced the asparagine inthe original heavy chain sequence.

MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSGADSGI(SEQ ID NO: 16)VVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKTSTYKREVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 9 Trastuzumab Light Chain Expression in Aspergillus

DNA of the integrative (i.e., it is designed to integrate into the hostgenomic DNA) expression plasmid pQ83 was prepared and transformed intoAspergillus niger var. awamori strain dgr246ΔGAP:pyr2-. This strain isderived from strain dgr246 P2 which has the pepA gene deleted, is pyrGminus and has undergone several rounds of mutagenesis and screening orselection for improved production of a heterologous gene product (Ward,M. et al., 1993, Appl. Microbiol. Biotech. 39:738-743 and referencestherein). To create strain dgr246ΔGAP:pyr2- the glaA (glucoamylase) genewas deleted in strain dgr246 P2 using exactly the same deletion plasmid(pΔGAM NB-Pyr) and procedure as reported by Fowler, T. et al (1990)Curr. Genet. 18:537-545. Briefly, the deletion was achieved bytransformation with a linear DNA fragment having glaA flanking sequencesat either end and with part of the promoter and coding region of theglaA gene replaced by the Aspergillus nidulans pyrG gene as selectablemarker. Transformants in which the linear fragment containing the glaAflanking sequences and the pyrG gene had integrated at the chromosomalglaA locus were identified by Southern blot analysis. This change hadoccurred in transformed strain dgr246ΔGAP. Spores from this transformantwere plated onto medium containing fluoroorotic acid and spontaneousresistant mutants were obtained as described by van Hartingsveldt, W. etal. (1987) Mol. Gen. Genet. 206:71-75. One of these, dgr246ΔGAP:pyr2-,was shown to be a uridine auxotroph strain which could be complementedby transformation with plasmids bearing a wild-type pyrG gene.

The Aspergillus transformation protocol was a modification of theCampbell method (Cambell et at. (1989). Curr. Genet. 16:53-56). Allsolutions and media were either autoclaved or filter sterilized througha 0.2 micron filter. Spores of A. niger var. awamori were harvested fromcomplex media agar (CMA) plates. CMA contained 20 g/l dextrose, 20 g/lDifcoBrand malt extract, 1 g/l Bacto Peptone, 20 g/l Bacto agar, 20 ml/lof 100 mg/ml arginine and 20 ml/l of 100 mg/ml uridine. An agar plug ofapproximately 1.5 cm square of spores was used to inoculate 100 mls ofliquid CMA (recipe as for CMA except that the Bacto agar was omitted).The flask was incubated at 37° C. on a shaker at 250-275 rpm, overnight.The mycelia were harvested through sterile Miracloth (Calbiochem, SanDiego, Calif., USA) and washed with 200 mls of Solution A (0.8M MgSO₄ in10 mM sodium phosphate, pH 5.8). The washed mycelia were placed in asterile solution of 300 mg of beta-D-glucanase (Interspex Products, SanMateo, Calif.) in 20 mls of solution A. This was incubated at 28° C. at200 rpm for 2 hour in a sterile 250 ml plastic bottle (Corning Inc,Corning, N.Y.). After incubation, this protoplasting solution wasfiltered through sterile Miracloth into a sterile 50 ml conical tube(Sarstedt, USA). The resulting liquid containing protoplasts was dividedequally amongst four 50 ml conical tubes. Forty ml of solution B (1.2 Msorbitol, 50 mM CaCl₂, 10 mM Tris, pH7.5) were added to each tube andcentrifuged in a table top clinical centrifuge (Damon IEC HN SIIcentrifuge) at ¾ speed for 10 minutes. The supernatant from each tubewas discarded and 20 mls of fresh solution B was added to one tube,mixed, then poured into the next tube until all the pellets wereresuspended. The tube was then centrifuged at ¾ speed for 10 minutes.The supernatant was discarded, 20 mls of fresh solution B was added, thetube was centrifuged for 10 minutes at ¾ speed. The wash occurred onelast time before resuspending the washed protoplasts in solution B at adensity of 0.5−1.0×10⁷ protoplasts/100 ul. To each 100 ul of protoplastsin a sterile 15 ml conical tube (Sarstedt, USA), 10 ul of thetransforming plasmid DNA was added. To this, 12.5 ul of solution C (50%PEG 4000, 50 mM CaCl₂, 10 mM Tris, pH 7.5) was added and the tube wasplaced on ice for 20 minutes. One ml of solution C was added and thetube was removed from the ice to room temperature and shaken gently. Twoml of solution B was added immediately to dilute solution C. Thetransforming mix was added equally to 3 tubes of melted MMS overlay (6g/l NaNO₃, 0.52 g/l KCl, 1.52 g/l KH₂PO₄, 218.5 g/l D-sorbitol, 1.0 ml/ltrace elements-LW, 10 g/l SeaPlaque agarose (FMC Bioproducts, Rook1 and,Maine, USA) 20 ml/l 50% glucose, 2.5 ml/l 20% MgSO₄.7H₂O, pH to 6.5 withNaOH) that were stored in a 45° C. water bath. Trace elements-LWconsisted of 1 g/l FeSO₄.7H₂O, 8.8 g/l ZnSO₄.7H₂O, 0.4 μl CuSO₄.5H₂O,0.15 g/l MnSO₄.4H₂O, 0.1 g Na₂B₄O₇.10H₂O, 50 mg/l (NH₄)₆Mo₇O₂₄.4H₂O, 250mls H₂O, 200 ul/l concentrated HCl. The melted overlays with thetransformation mix were immediately poured onto 3 MMS plates (same asMMS overlay recipe with the exception of 20 g/l of Bacto agar instead of10 g/l of SeaPlaque agarose) that had been supplemented with 200ul/plate of 100 mg/ml of arginine added directly on top of the agarplate. After the agar solidified, the plates were incubated at 37° C.until transformants grew.

The sporulating transformants were picked off with a sterile toothpickonto a plate of minimal media+glucose (MM). MM consisted of 6 g/l NaNO₃,0.52 g/l KCl, 1.52 g/l KH₂PO₄, 1 ml/l Trace elements-LW, 20 g/l Bactoagar, pH to 6.5 with NaOH, 25 ml/l of 40% glucose, 2.5 ml/l of 20%MgSO₄.7H₂O and 20 ml/l of 100 mg/ml arginine. Once the transformantsgrew on MM they were transferred to CMA plates.

A 1.5 cm square agar plug from a plate culture of each transformant wasadded to 50 mls, in a 250 ml shake flask, of an inoculum medium calledCSL+fructose 100 g/1 corn steep liquor (50% solids, National), 1 g/lNaH₂PO₄.H₂O, 0.5 μl MgSO₄, 100 μl maltose, 10 g/l glucose, 50 g/lfructose, 3 m1/l Mazu DF60-P (Mazur Chemicals, Gurnee, Ill., USA), pH to5.8 with NaOH. Flasks were incubated at 37° C., 200 rpm, for 2 days.Five ml of the 2 day old medium were inoculated into 50 ml of productionmedium called Promosoy special. This medium had the followingcomponents: 70 g/l sodium citrate, 15 g/l (NH₄)₂ SO₄, 1 g/l NaH₂PO₄.H₂O,1 g/l MgSO₄, 1 ml Tween 80, pH to 6.2 with NaOH, 2 ml/l Mazu DF60-P, 45g/l Promosy 100 (Central Soya, Fort Wayne, Ind.), 120 g/l maltose. Theproduction media flasks were incubated at 30° C., 200 rpm for 5 days andsupernatant samples were harvested.

Samples of culture supernatant were mixed with an appropriate volume of2× sample loading buffer and subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using precast gelsaccording to the manufacturers instructions (The NuPAGE Bis-TrisElectrophoresis System from Invitrogen Corporation, Carlsbad, Calif.).The gels were either stained for protein with Coomassie Brilliant Bluestain or the protein was transferred to membrane filters by Westernblotting (Towbin et al., 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354).Human kappa light chain was visualized on Western blots by sequentialtreatment with goat anti-human kappa light chain (bound and free)antibody and rabbit anti-goat IgG conjugated with horse radishperoxidase (HRP) followed by HRP color development by incubation withH₂O₂ and 4-chloro-l-napthol.

Transformants which produced trastuzumab light chain were identified bythe appearance of extra protein bands compared to supernatant from theuntransformed parental strain. The sizes and identities of these bandswere as follows. A 25 kD band corresponding to the trastuzumab lightchain which had been released from the glucoamylase-light chain fusionprotein. A band with an apparent molecular weight of approximately 58 kDcorresponding to the catalytic core and linker region of glucoamylasewhich had been released from the glucoamylase-light chain fusionprotein. A band with an apparent molecular weight of approximately 85 kDcorresponding to the glucoamylase-light chain fusion protein which hadnot been cleaved into separate glucoamylase and light chain proteins.The identities of the light chain bands were confirmed by Westernanalysis. The Western analysis employed an anti-human κ antibody todetect both the free light chain and the glucoamylase-light chain fusionprotein. Quantification of the light chain in culture supernatants wasperformed by enzyme-linked immunosorption assays (ELISA). The best lightchain expression strain was spore purified and was designated Q83-35-2.This strain produced approximately 1.5 g/l of trastuzumab light chain (κchain) in shake flask culture according to ELISA. The ELISA wasperformed using goat anti-human κ (bound and free) antibody as a captureantibody coating the wells of microtiter plates. After addingappropriately diluted culture supernatant, incubation, and then washingthe wells, the bound light chain from the supernatant was detected byaddition of an goat anti human kappa (bound and free) antibodyconjugated with horse radish peroxidase (HRP) followed by a colordevelopment reaction. A serial dilution of known concentration for humanK light chain was used to produce a standard for quantificationpurposes.

Example 10 Improved Cleavage of the Glucoamylase-Light Chain FusionProtein

As indicated above, some of the trastuzumab light chain remainedattached to glucoamylase when secreted by Aspergillus nigertransformants containing pQ83. It was estimated that approximately60-75% of the secreted light chain was attached to glucoamylase. Thisindicated that the KEX2 site between glucoamylase and the light chainwas not efficiently cleaved by the KEX2 protease. In order to determineif the site of cleavage was as predicted (i.e., immediately after the KRresidues of the KEX2 cleavage site) the N-terminus of the free lightchain from transformant Q83-35-2 was determined. Proteins in culturesupernatant samples were separated by SDS-PAGE and were blotted onto apolyvinylidene difluoride (PVDF) membrane using a Novex transfer cell(Invitrogen Corporation, Carlsbad, Calif.) and transfer bufferconsisting of 12 mM Tris base, 96 mM glycine, 20% methanol, 0.01% SDS,pH8.3. The transfer was run at 20 V for 90 minutes. The membrane wasrinsed three times for 30 minutes each in distilled water and stainedwith Coomassie Brilliant Blue R-250. The portion of the membrane withthe 25 kD light chain band was excised and the N-terminal sequence wasdetermined by Edman degradation. The data indicated that the populationof light chain molecules had a mixture of N-termini, the dominantsequences were DIQM and KRDI and these were present in approximatelyequal amounts. This result-demonstrates that some of theglucoamylase-light chain fusion proteins were cleaved at the expectedposition immediately after the KEX2 cleavage site but that approximatelyhalf of the cleaved fusion proteins had been cleaved at a position tworesidues towards the N-terminus.

In order to improve cleavage of the fusion protein we altered theposition on the glucoamylase linker region to which the light chain wasattached. Additionally, the amino acid sequence at the junction betweenglucoamylase and light chain was varied.

The expression plasmid used for these experiments was based on the samevector as pGAKHi+, a glucoamylase-hirulog expression vector which isdescribed in detail in WO 9831821. The hirulog-encoding region of thisplasmid, which is situated between unique NheI and BstEII restrictionendonuclease recognition sites, was replaced by light chain-encodingDNA. NheI cuts the plasmid within the coding region for the glucoamylaselinker region and BstEII cuts 5 of the glucoamylase terminator region.Using techniques known in the art the DNA sequence encoding the completelight chain was amplified by PCR using the following pair of primers.5-CCGCTAGCAAGCGTGATATCCAG-3 (SEQ ID NO:17) was the forward primer and5-CCGGTGACCGGATCAACACTCTCCC-3 (SEQ ID NO:18) was the reverse primer.These primers added NheI and BstEII recognition sites at the 5 and 3ends of the light chain DNA respectively. The light chain DNA was theninserted into the vector to create a plasmid identical to pGAKHi+ butwith the light chain DNA replacing the hirulog-encoding region to createpQ87. This plasmid contained the Aspergillus niger pyrG gene as aselectable marker for transformation into Aspergillus or other fungi.The Aspergillus niger var awamori g/aA (glucoamylase) promoter and A.niger glaA terminator were included to control expression of the openreading frame which includes the light chain encoding DNA. This plasmidwas designed for the expression of the fusion protein shown belowconsisting of the glucoamylase signal sequence, prosequence, catalyticdomain and linker region up to amino acid number 498 (a serine) ofmature glucoamylase (Nunberg, J. H. et al., 1984, Mol. Cell. Biol.4:2306-2315), followed by amino acids KR (underlined below) and then bythe mature light chain.

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 19)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEWDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The amino acid sequence on either side of the KR residues (kexB cleavagesite) was altered in a series of plasmids. To construct each new lightchain expression plasmid, the following forward primers were used, eachin combination with the same reverse primer as described for pQ87, toamplify the light chain DNA fragment in a PCR reaction.5-CCGCTAGCCAGAAGCGTGATATCCAGA-3 (SEQ ID NO:20) was the forward primerfor pQ88; 5-CCGCTAGCCTCAAGCGTGATATCCAG-3 (SEQ ID NO:21) was the forwardprimer for pQ90; 5-CCGCTAGCATCTCCAAGCGTGATATCCAG-3 (SEQ ID NO:22) wasthe forward primer for pQ91; 5-CCGCTAGCAACGTGATCTCCAAGCGTGATATCCAG-3(SEQ ID NO:23) was the forward primer for pQ94;5-CCGCTAGCGTGATCTCCAAGCGTGATATCCAG-3 (SEQ ID NO:24) was the forwardprimer for pQ95; and 5-CCGCTAGCATCTCCAAGCGTGGCGGTGGCGATATCCAGATGACCCAG-3(SEQ ID NO:25) was the forward primer for pQ96; The PCR fragment wasthen digested with restriction enzymes NheI and BstEII and inserted intothe expression vector as for pQ87. In pQ88 and pQ90, an amino acid wasinserted at the amino-terminal side of the KR residues which had beenshown to be accepted at this position for cleavage of synthetic peptidesby yeast KEX2 and A. niger KexB (Brenner, C. and Fuller, R. S., 1992,Proc. Natl. Acad. Sci. USA 89:922-926; Jalving, R. et al., 2000, Appliedand Environmental Microbiology 66:363-368). In pQ91, pQ94 and pQ95, two,four or three residues respectively from the 6 amino acid propeptide ofglucoamylase (which ends with KR and is cleaved by KEX2 protease) wereplaced on the amino-terminal side of the KR residues. Residues from theglucoamylase propeptide sequence have been placed in this position inglucoamylase fusion proteins by others (Spencer, J. A. et al., 1998,European Journal of Biochemistry 258:107-112; Broekhuijsen, M. P. etal., 1993, Journal of Biotechnology 31:135-145). In pQ96, three glycineresidues were placed on the carboxyl side of the KR residues as had beenemployed by (Spencer, J. A. et al., 1998, European Journal ofBiochemistry 258:107-112). For each plasmid the amino acid sequence ofthe encoded glucoamylase-light chain fusion protein is shown below andthe variable region around the KEX2 cleavage site (KR) is underlined.

Glucoamylase-light chain fusion protein encoded by pQ88:

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 26)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASQKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Glucoamylase-light chain fusion protein encoded by pQ90:

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 27)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASLKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Glucoamylase-light chain fusion protein encoded by pQ91:

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 28)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASISKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Glucoamylase-light chain fusion protein encoded by pQ94:

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 29)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEWDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSWPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASNVISKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Glucoamylase-light chain fusion protein encoded by pQ95:

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 30)ADSGIWASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASVISKRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Glucoamylase-light chain fusion protein encoded by pQ96:

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 31)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASISKRGGGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

DNA of the expression plasmids pQ87, pQ88, pQ90, pQ91, pQ94, pQ95 andpQ96 were prepared and each was transformed individually intoAspergillus niger var. awamori strain dgr246ΔGAP:pyr2-. Thetransformants obtained were cultured in shake flasks in Promosoy specialmedium and the secreted proteins were visualized by SDS-PAGE andCoomassie Brilliant Blue staining. Cleavage of the glucoamylase-lightchain fusion protein was assessed by examining the relative amounts ofthe 25 kD band corresponding to the trastuzumab light chain which hadbeen released from the fusion protein, the 58 kD band corresponding tothe catalytic core and linker region of glucoamylase which had beenreleased from the fusion protein and the approximately 85 kD bandcorresponding to the glucoamylase-light chain fusion protein which hadnot been cleaved into separate glucoamylase and light chain proteins. Inaddition, the N-terminus of the released light chain was determined insome instances.

The extent of cleavage of the glucoamylase-light chain fusion proteinwas apparently unchanged, at approximately 25 to 40%, in A. nigertransformants with the expression vectors pQ87, pQ88 or pQ90 compared totransformant Q83-35-2.

In contrast, approximately 90% of the glucoamylase-light chain fusionprotein was cleaved in transformants with expression vectors pQ91, pQ94and pQ95. The amino terminus of the free light chain in the supernatantsof one transformant obtained with each of pQ91 and pQ94 was determinedand a single dominant sequence of DIQMT (SEQ ID NO:41) was observed.This demonstrated that not only the extent of cleavage was improved intransformants with these expression vectors but also frequency at whichthe fusion protein was cleaved at the expected KEX2 site, i.e., theaccuracy or fidelity of the cleavage had been improved.

100% of the glucoamylase-light chain fusion protein was apparentlycleaved in transformants with expression vector pQ96. The amino terminusof the free light chain in the supernatant of one transformant obtainedwith pQ96 was determined and a single dominant sequence of GGGDI (SEQ IDNO:42) was observed.

Example 11 Trastuzumab Heavy Chain Expression in Aspergillus

DNA of the integrative expression plasmid pCL1 was prepared andtransformed into Aspergillus niger var. awamori strain dgr246ΔGAM.Transformants were cultured in liquid medium in shake flasks as above.In some experiments, the trastuzumab heavy chain was specificallyprecipitated from the supernatant by incubation with protein A-sepharosebeads (Amersham Pharmacia) which has specific affinity for the heavychain of IgG. The beads were pre-washed in SDS-PAGE running buffer andwere further washed in this buffer after incubation with heavy chain andbefore being resuspended in SDS-PAGE sample buffer, heating at 70° C.for 10 minutes prior and loading on a polyacrylamide gel. Samples ofsupernatant, or of material precipitated from supernatant with proteinA-sepharose, were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) under reducing conditions followed by Coomassie BrilliantBlue staining of the protein bands or blotting to nylon membranes forWestern analysis. Trastuzumab heavy chain was visualized on Westernblots by sequential treatment with goat anti-human IgG-Fc antibody andrabbit anti-goat IgG conjugated with horse radish peroxidase (HRP)followed by HRP color development by incubation with H₂O₂ and4-chloro-1-napthol. Transformants which produced trastuzumab heavy chainwere identified by the appearance of extra protein bands compared tosupernatant from the untransformed parental strain. The sizes andidentities of these bands were as follows. A pair of bands, one at 50 kD(which has the same mobility on SDS-PAGE as the secreted alpha-amylaseof Aspergillus niger), and one at approximately 53 KD, bothcorresponding to the trastuzumab heavy chain which had been releasedfrom the glucoamylase-heavy chain fusion protein (the appearance of freeheavy chain of two different sizes is explained below). A band with anapparent molecular weight of 58 kD corresponding to the catalytic coreand linker region of glucoamylase which had been released from theglucoamylase-heavy chain fusion protein. A band with an apparentmolecular weight of 116 kD corresponding to the glucoamylase-heavy chainfusion protein which had not been cleaved into separate glucoamylase andheavy chain proteins. The best heavy-chain expressing transformantproduced approximately 0.33 g per liter of culture supernatant oftrastuzumab gamma heavy chain according to ELISA. The ELISA wasperformed using goat anti-human IgG-Fc antibody as a capture antibodycoating the wells of microtiter plates. After adding appropriatelydiluted culture supernatant, incubation, and then washing the wells, thebound heavy chain from the supernatant was detected by addition of goatanti human IgG-Fc antibody conjugated with HRP followed by a colordevelopment reaction. A serial dilution of known concentration of humanIgG was used to produce a standard for quantification purposes.

Example 12 Trastuzumab Heavy and Light Chain Expression in Aspergillus

Three different transformation strategies were used to constructAspergillus transformants which produced both heavy and light chains ofthe trastuzumab antibody.

Strain construction by co-transformation. The expression plasmids pQ83and pCL1 were mixed and transformed into Aspergillus niger var. awamoristrain dgr246ΔGAM. Neither of these plasmids has a fungal origin ofreplication and would be expected to integrate into the Aspergilluschromosomal DNA at one or more sites. Transformants were cultured inshake flasks as described above and light and heavy chain production wasevident from SDS-PAGE and Western analysis. A mix of the same lightchain and heavy chain bands were observed as seen in the transformantswhich produced only light chain or only heavy chain. Up to approximately0.3 g/l assembled IgG was measured by ELISA in shake flask cultures ofthe best transformant (1-LC/HC-3). The ELISA was performed using goatanti-human IgG-Fc antibody as a capture antibody coating the wells ofmicrotiter plates. After adding appropriately diluted culturesupernatant, incubation; and then washing the wells, the bound IgG1 fromthe supernatant was detected by addition of an goat anti human κ (boundand free) antibody conjugated with HRP followed by a color developmentreaction. By employing this combination of capture and detectionantibodies in the ELISA, only assembled IgG1 would be measured whereasfree light chain and heavy chain would not be measured. A serialdilution of known concentration of purified human IgG was used toproduce a standard for quantification purposes. The capture anddetection antibodies were reversed in some experiments so that thecapture antibody was anti human K antibody and the detection antibodywas anti-human IgG-Fc antibody conjugated with HRP. Results werecomparable with either combination of antibodies.

Transformants were also obtained by co-transformation with theexpression plasmids pQ83 and pCL5. These plasmids were mixed andtransformed into Aspergillus niger var. awamori strain dgr246ΔGAM. Up to0.9 g/l assembled IgG was measured by ELISA in shake flask cultures ofthe best transformant (2-LC/HC-38).

Strain construction using a replicating plasmid. The plasmids pQ83, pCL1and pHELP1 were mixed and transformed into Aspergillus niger var.awamori strain dgr246ΔGAM. The plasmid pHELP1 (Gems, D. and Clutterbuck,A. J., 1993, Curr. Genet. 24:520-524) includes an Aspergillus nidulanssequence, AMA1, which confers autonomous replication in aspergillusstrains. Based on previous results (Gems, D. and Clutterbuck, A. J.,1993, Curr. Genet. 24:520-524) it would be expected that the plasmidswould recombine with one another and form a large replicating plasmidthat contains elements of all three plasmids. Transformants werecultured in shake flasks and analyzed for expression of the trastuzumabheavy and light chains by SDS-PAGE and Western analysis. A mix of thesame light chain and heavy chain bands were observed as seen in thetransformants which produced only light chain or only heavy chain.Assembled IgG1 was assayed by ELISA. Up to 0.26 g/l assembled IgG wasmeasured by ELISA in shake flask cultures.

Strain construction by two sequential transformations. The integrativeplasmid pCL1 (heavy chain expression plasmid) was used to transformstrain Q83-35-2, the best light chain producing strain identified above.Transformants were cultured in shake flasks and analyzed for expressionof the trastuzumab heavy and light chains by SDS-PAGE and Westernanalysis. A mix of the same light chain and heavy chain bands wereobserved as seen in the transformants which produced only light chain oronly heavy chain. Assembled IgG1 was assayed by ELISA. Up to 0.19 g/lassembled IgG was measured by ELISA in shake flask cultures.

In some experiments, the trastuzumab heavy chain and associated lightchain was specifically precipitated from the supernatant by incubationwith Protein A-Sepharose 4 Fast Flow beads (Amersham Pharmacia,Piscataway, N.J.) as above. Purification of the heavy chain andassociated light chain was also performed using affinity chromatographyon a HiTrap Protein A HP chromatography column (Amersham Pharmacia,Piscataway, N.J.) following the manufacturers protocol. FIG. 8 shows theresults of SDS-PAGE under reducing conditions and with CoomassieBrilliant Blue staining of samples which had been purified by protein Achromatography. The bands observed for transformant 1-LC/HC-3 wereidentified as the light chain (25 kDa), non-glycosylated andglycosylated forms of the heavy chain (50 and 53 kDa),glucoamylase-light chain fusion protein (85 kDa) and glucoamylase-heavychain fusion (116 kDa). The fact that light chain was co-purified withheavy chain by Protein A affinity chromatography (which is specific forheavy chain) demonstrated that the antibody was assembled. The fact thatboth glucoamylase-heavy chain and glucoamylase-light chain fusionproteins co-purified by Protein A affinity chromatography demonstratedthat the antibody assembled with glucoamylase attached.

FIG. 9 shows the results of SDS-PAGE (NuPAGE Tris-AcetateElectrophoresis System from Invitrogen Corporation, Carlsbad, Calif.)under non-reducing conditions and with Coomassie Brilliant Blue stainingof samples which had been purified by protein A chromatography. Themajor bands observed for transformant 1-LC/HC-3 were identified asassembled IgG1 (150 kDa), assembled IgG1 with one molecule ofglucoamylase attached (˜200 kDa) and assembled IgG1 with two moleculesof glucoamylase attached (˜250 kDa).

In order to understand why the two forms of the free heavy chain (i.e.,that heavy chain which was released from the glucoamylase-heavy chainfusion protein) were produced differing in apparent molecular weight byapproximately 3 kD, the following experiments were performed. Thetrastuzumab produced by Aspergillus was purified by protein A affinitychromatography. Samples of the purified trastuzumab were incubated for 1hour in the presence or absence of 35 ug ofendo-β-N-acetylglucosaminidase H (endo H) which is able to cleave highmannose type N-linked glycosylation from proteins leaving a singleN-acetylglucosamine sugar attached to the asparagine of the protein.These samples were analyzed by SDS-PAGE under reducing conditionsfollowed by Coomassie Brilliant Blue staining for proteins or stainingwhich is specific for glycoproteins (GelCode Glycoprotein Staining Kitfrom Pierce, Rockford, Ill. used according to the manufacturesinstructions). The upper of the two bands of free heavy chain wasgreatly reduced in intensity by treatment with endo H. Only the upperfree heavy chain band was stained with GelCode stain and this band wasno longer visible with GelCode staining after endo H treatment. Theseobservations indicate that the upper of the two free heavy chain bandsrepresents heavy chain with N-linked high mannose glycan attached andthat endo H treatment is able to remove this glycan.

It was possible to purify and separate the free IgG1 from theglucoamylase-IgG1 fusion proteins. The method used for purification washydrophobic charge induction chromatography as described in co-pendingapplication U.S. Ser. No. 60/411,537 filed Sep. 18, 2002, entitled“Protein Purification”. Firstly, fungal cells were removed from culturebroth by filtration through Miracloth (Calbiochem, San Diego, Calif.).The filtered broth was concentrated approximately seven-fold bytangential ultrafiltration. Using a circulating pump, the broth waspressurized and flowed across a membrane made of regenerated cellulosewith a 30,000 molecular weight cutoff (Prep/Scale™ TFF, Millipore). Toremove particulates, the concentrate was centrifuged at 25,000 timesgravity for 15 minutes, and the supernatant was filtered through aseries of membranes, with each membrane having a smaller pore size thanthe previous, ending with 0.2-micrometer pore size. IgG1 was purifiedfrom supernatant using hydrophobic charge induction chromatography(HCIC) This was performed with the aid of a high performance liquidchromatographic system (AKTA™ explorer 10, Amersham Biosciences). HCICprovided an ability to separate antibody molecules from othersupernatant proteins and from glucoamylase-fusion proteins. It wascarried out using a column containing MEP HyperCel® (CiphergenBiosystems) media. The column was equilibrated with 50 mM Tris, 200 mMNaCl, and pH 8.2 buffer. Supernatant, adjusted to pH 8.2, was applied tothe column at a linear flow rate of 100 cm/h. After washing with fivecolumn volumes (5 CV) of equilibration buffer, bound molecules wereeluted by incrementally decreasing the pH. Two CV of each of thefollowing buffers were delivered to the column at 200 cm/h, in the orderlisted: 100 mM sodium acetate, pH 5.6; 100 mM sodium acetate, pH 4.75;100 mM sodium acetate, pH 4.0; and 100 mM sodium citrate, pH 2.5. FreeIgG1 eluted within the pH range 4.5-5.5 and was immediately neutralizedwith 1 M Tris and pH 8.2 buffer. The purity of the antibody exiting thecolumn was assessed by SDS-PAGE.

Example 13 A Glycosylated Trastuzumab Expression in Aspergillus

The plasmid pCL3 (the expression vector for the aglycosylated mutantform of the heavy chain) was used to transform strain Q83-35-2, the bestlight chain producing strain identified above. Transformants werecultured in shake flasks. Both light chain and heavy chain expressionwas evident from SDS-PAGE after precipitation of the heavy chain withprotein A-sepharose beads or after purification by protein A affinitychromatography. A mix of the same light chain and heavy chain bands wereobserved on SDS-PAGE under reducing conditions as seen in transformant1-LC/HC-3 except that only a single band of free heavy chain at 50 kDwas observed (strain 1-HCΔ4 in FIG. 8). A similar pattern of bands wereobserved on SDS-PAGE under non-reducing conditions as seen intransformant 1-LC/HC-3 (strain 1-HCΔ4 in FIG. 9). Fully assembled IgG1was measured by ELISA. 0.1 g/l of aglycosylated trastuzumab was producedin shake flask cultures by the best transformant.

Example 14 Trastuzumab Fab′ Fragment Expression in Aspergillus

The plasmid pCL2 (the expression vector for the Fd′ fragment of thetrastuzumab heavy chain) was used to transform strain Q83-35-2, the bestlight chain producing strain identified above. Transformants werecultured in shake flasks. Assembled Fab′ was measured by ELISA. Twotransformants were studied in more detail; 1-Fab-1 and 1-Fab-12. 1.2 g/lFab′ was produced in shake flask cultures by the best transformant(strain 1-Fab-12). Expression of the Fab′ fragment of the trastuzumabwas evident from SDS-PAGE after precipitation of the heavy chain withprotein A-sepharose beads or after purification by protein A affinitychromatography. SDS-PAGE under reducing conditions showed a band atapproximately 25 kDa representing both the light chain and Fd′ chains aswell as a band at approximately 85 kDa representing both theglucoamylase-light chain and glucoamylase-Fd′ chain fusion proteins(strain 1-Fab-1 in FIG. 8). The major bands observed on SDS-PAGE undernon-reducing conditions were one at approximately 50 kDa representingthe assembled Fab′ and one at approximately 100 kDa representing Fab′with a single glucoamylase molecule attached (strain 1-Fab-1 in FIG. 9).A fainter band at approximately 150 kDa may represent Fab′ withglucoamylase molecules attached to both the light chain and the Fd′chain. It is of interest to determine if Fab′ produced by A. niger cancovalently dimerize through the free cysteines near the carboxylterminus of the Fd′ chain to form F(ab′)₂. The size of F(ab′)₂ would beapproximately 100 kDa and would therefore run at the same position asFab′ with a single glucoamylase molecule attached on non-reducingSDS-PAGE. Similarly, the size of F(ab′)₂ with one glucoamylase attachedwould be approximately 150 kDa and would therefore run at the sameposition as Fab′ with two glucoamylase molecules attached. However, thehigher molecular weight band at approximately 200 kDa observed forstrain 1-Fab-1 in FIG. 9 is best explained as representing F(ab′)₂ withtwo glucoamylase molecules attached.

To confirm that F(ab′)₂ was secreted by transformant 1-Fab-12 thesecreted antibody fragments were purified to separate the Fab′ andF(ab′)₂ from the glucoamylase-Fab′ and glucoamylase-F(ab′)₂ fusionproteins. The method used for purification was hydrophobic chargeinduction chromatography as described in co-pending application U.S.Ser. No. 60/411,537 filed Sep. 18, 2002, entitled “Protein Purification”followed by size exclusion chromatography. Firstly, fungal cells wereremoved from culture broth by filtration through Miracloth (Calbiochem,San Diego, Calif.). The filtered broth was concentrated approximatelyseven-fold by tangential ultrafiltration. Using a circulating pump, thebroth was pressurized and flowed across a membrane made of regeneratedcellulose with a 30,000 molecular weight cutoff (Prep/Scale™ TFF,Millipore). To remove particulates, the concentrate was centrifuged at25,000 times gravity for 15 minutes, and the supernatant was filteredthrough a series of membranes, with each membrane having a smaller poresize than the previous, ending with 0.2-micrometer pore size. Fab′(monomer and dimer) antibody fragments were purified from supernatantusing a combination of hydrophobic charge induction chromatography(HCIC) and size exclusion chromatography (SEC). Each of these methodswas performed with the aid of a high performance liquid chromatographicsystem (AKTA™ explorer 10, Amersham Biosciences). HCIC provided anability to separate antibody molecules from other supernatant proteinsand from glucoamylase-fusion proteins. SEC served to separate Fab fromF(ab′)₂. HCIC was carried out using a column containing MEP HyperCel®(Ciphergen Biosystems) media. The column was equilibrated with 50 mMTris, 200 mM NaCl, and pH 8.2 buffer. Supernatant, adjusted to pH 8.2,was applied to the column at a linear flow rate of 100 cm/h. Afterwashing with five column volumes (5 CV) of equilibration buffer, boundmolecules were eluted by incrementally decreasing the pH. Two CV of eachof the following buffers were delivered to the column at 200 cm/h, inthe order listed: 100 mM sodium acetate, pH 5.6; 100 mM sodium acetate,pH 4.75; 100 mM sodium acetate, pH 4.0; and 100 mM sodium citrate, pH2.5. Fab′ and F(ab′)₂ eluted within the pH range 4.5-5.5 and wereimmediately neutralized with 1 M Tris and pH 8.2 buffer. A HiLoad™ 26/60column with Superdex 200™ Prep Grade media (Amersham Biosciences) wasused for SEC. The flow rate was kept at 17 cm/h. After equilibrating thecolumn with 20 mM sodium acetate, 136 mM NaCl, and pH 5.5 buffer; a6.5-mL sample was driven through the column with 1 CV of equilibrationbuffer. The purity of the antibody exiting the column was assessed bySDS-PAGE.

On SDS-PAGE under reducing conditions the Fd′ and light chains of theHCl-purified Fab′ and F(ab′)₂ both run as bands of 25 kDa (FIG. 10).Under these conditions it is clear that no glucoamylase-light chain orglucoamylase-Fd′ fusion proteins are present in the purified samplesbecause these would run as a band of approximately 50 kDa. On SDS-PAGEunder non-reducing conditions it is clear that F(ab′)₂ is present in thepurified samples (Fraction A5 in FIG. 10) because this runs as a band ofapproximately 100 kDa compared to the 50 kDa of Fab′ (Fraction B7 inFIG. 10).

Example 15 Assays to Demonstrate that Trastuzumab Made in Aspergillus isFunctional (it Binds to and Inhibits Proliferation of her2 ExpressingBreast Cancer Cells)

The effect of the trastuzumab produced by Aspergillus transformant1-LC/HC-3 was compared to that of commercial trastuzumab (Herceptin,Genentech, South San Francisco, Calif.) on the proliferation of a humanbreast adenocarcinoma cell line, SK-BR-3 (ATCC number: HTB-30), whichexpresses high levels of HER2. In order to assay proliferation of thecells in 96 well microtiter plates the “CellTiter 96 Aqueous OneSolution Cell Proliferation Assay” (Promega Corporation, Madison, Wis.)was used according to the manufacturers instructions. SK-BR-3 cells wereplated at 1800 cells per well and allowed to adhere for 6 hours prior toantibody addition then assayed 72 hours later. Protein A purified IgG1from transformant 1-LC/HC-3 was tested for anti-proliferative effects onSK-BR-3 cells relative to Herceptin and untreated cells. As a controlcell line A-431 cells (ATCC number: CRL-1555) were used. A-431 is humanepidermoid carcinoma that expresses high levels of the EGF receptor andlow levels of HER2. Herceptin should have little or noanti-proliferative effect on this cell line. Data is presented as thepercent proliferation (mean of triplicate wells) relative to untreatedcells (see FIG. 11). These results are in excellent agreement with thereported anti-proliferative effects of trastuzumab on the SK-BR-3 cellline (Carter, P. et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-4289)and demonstrate that the antibody purified from culture supernatant oftransformant 1-LC/HC-3 is assembled and functional in its ability tobind to the specific antigen, HER2.

Example 16 Production of Hu1D10 Antibody in Aspergillus

Expression vectors were constructed in the same manner as described inExample 10 to allow the production of the light and heavy chains ofHu1D10 antibody (of the IgG1κ subclass; Kostelny, S. a. et al., 2001,Int. J. Cancer 93:556-565) in Aspergillus niger. The cDNA encodingHu1D10 was modified by site directed mutagenesis to remove internalBstEII sites. PCR primers were designed to amplify and add NheI sites atthe 5 end, add BstEII sites at the 3 end and to add specific codons atthe 5 ends.

Two forms of the cDNA encoding the Hu1D10 light chain were generatedwhich varied at the 5 end sequence. The cDNA sequence encoding the Q101form of Hu1D10 light chain was as follows. The nucleotides representedin lower case are those added by the PCR primers.

     GctagcatctccaagcgcGACATCCAGATGACTCAGTCTCCATCTTCTCTATCTGCAT(SEQ ID NQ: 32)CTGTGGGAGACAGGGTCACAATCACATGTCGAGCAAGTGAAAATATTTACAGTTATTTAGCATGGTACCAGCAGAAACCTGGAAAAGCTCCTAAGCTCCTGGTCTCTAATGCTAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCAAACAGTTTACTCTGACAATCAGCAGCCTGCAGCCTGAAGATTTTGCTACTTATTACTGTCAACATCATTATGGTAATTCGTACCCGTTCGGACAGGGGACCAAACTGGAAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGggtgacc.

The cDNA sequence encoding the Q100 form of Hu1D10 light chain was asfollows. The nucleotides represented in lower case are those added bythe PCR primers.

     GctagcatctccaagcgcggtggcggaGACATCCAGATGACTCAGTCTCCATCTTCTCTA(SEQ ID NO: 33)TCTGCATCTGTGGGAGACAGGGTCACAATCACATGTCGAGCAAGTGAAAATATTTACAGTTATTTAGCATGGTACCAGCAGAAACCTGGAAAAGCTCCTAAGCTCCTGGTCTCTAATGCTAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCAAACAGTTTACTCTGACAATCAGCAGCCTGCAGCCTGAAGATTTTGCTACTTATTACTGTCAACATCATTATGGTAATTCGTACCCGTTCGGACAGGGGACCAAACTGGAAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGggtgacc.

The above amplified light chain cDNAs could then be inserted into theAspergillus expression vector to create pQ101 or pQ100. The Aspergillusniger var awamori glaA (glucoamylase) promoter and A. niger glaAterminator were present in the plasmid to control expression of the openreading frame which included the light chain encoding cDNA.

Plasmid pQ101 was designed for the expression of a fusion protein withthe amino acid sequence shown below and consisting of the glucoamylasesignal sequence, prosequence, catalytic domain and linker region up toamino acid number 498 (a serine) of mature glucoamylase (Nunberg, J. H.et al., 1984, Mol. Cell. Biol. 4:2306-2315), followed by amino acidsISKR (underlined below) and then by the mature Hu1D10 light chain. Thisplasmid did not include a selectable marker for Aspergillustransformation.

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 34)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASISKRDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVSNAKTLAEGVPSRFSGSGSGKQFTLTISSLQPEDFATYYCQHHYGNSYPFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Plasmid pQ100 was designed for the expression of a fusion protein withthe amino acid sequence shown below and consisting of the glucoamylasesignal sequence, prosequence, catalytic domain and linker region up toamino acid number 498 (a serine) of mature glucoamylase (Nunberg, J. H.et al. 1984 Mol. Cell. Biol. 4:2306-2315), followed by amino acidsISKRGGG (underlined below) and then by the mature Hu1D10 light chain.This plasmid also contained the A. niger pyrG gene as a selectablemarker for Aspergillus transformation.

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 35)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASISKRGGGDIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVSNAKTLAEGVPSRFSGSGSGKQFTLTISSLQPEDFATYYCQHHYGNSYPFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Two forms of the cDNA encoding the Hu1D10 heavy chain were generatedwhich varied at the 5 end sequence. The cDNA sequence encoding the CL17form of Hu1D10 light chain was as follows. The nucleotides representedin lower case are those added by the PCR primers.

     gctagcatctccaagcgcCAGGTGCAGCTGCAGGAGTCAGGACCAGGCCTAGTGAA(SEQ ID NO: 36)GCCCTCAGAGACTCTGTCCCTAACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAATGGATCGGAGTGAAATGGAGTGGTGGGTCGACAGAATATAATGCAGCTTTCATATCCAGACTGACCATCAGCAAGGACACCTCCAAGAACCAAGTTTCCCTTAAACTGAACAGTCTGACCGCTGCTGACACAGCCGTGTACTACTGTGCCAGAAATGATAGATATGCTATGGACTACTGGGGTCAAGGAACTCTAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAggtgacc.

The cDNA sequence encoding the CL16 form of Hu1D10 heavy chain was asfollows. The nucleotides represented in lower case are those added bythe PCR primers.

     gctagcatctccaagcgcggtggcggaCAGGTGCAGCTGCAGGAGTCAGGACCAGGCCT(SEQ ID NO: 37)AGTGAAGCCCTCAGAGACTCTGTCCCTAACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAATGGATCGGAGTGAAATGGAGTGGTGGGTCGACAGAATATAATGCAGCTTTCATATCCAGACTGACCATCAGCAAGGACACCTCCAAGAACCAAGTTTCCCTTAAACTGAACAGTCTGACCGCTGCTGACACAGCCGTGTACTACTGTGCCAGAAATGATAGATATGCTATGGACTACTGGGGTCAAGGAACTCTAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACAGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAggtgacc.

The above amplified heavy chain cDNAs could then be inserted into theAspergillus expression vector to create pCL17 or pQCL16. The Aspergillusniger var awamori glaA (glucoamylase) promoter and A. niger glaAterminator were present in the plasmid to control expression of the openreading frame which included the heavy chain encoding DNA.

Plasmid pCL17 was designed for the expression of a fusion protein withthe amino acid sequence shown below and consisting of the glucoamylasesignal sequence, prosequence, catalytic domain and linker region up toamino acid number 498 (a serine) of mature glucoamylase (Nunberg, J. H.et al., 1984, Mol. Cell. Biol. 4:2306-2315), followed by amino acidsISKR (underlined below) and then by the mature Hu1D10 heavy chain. Thisplasmid also contained the A. niger pyrG gene as a selectable marker forAspergillus transformation.

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 38)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASISKRQVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWVRQSPGKGLEWIGVKWSGGSTEYNAAFISRLTISKDTSKNQVSLKLNSLTAADTAVYYCARNDRYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Plasmid pCL16 was designed for the expression of a fusion protein withthe amino acid sequence shown below and consisting of the glucoamylasesignal sequence, prosequence, catalytic domain and linker region up toamino acid number 498 (a serine) of mature glucoamylase (Nunberg, J. H.et al., 1984, Mol. Cell. Biol. 4:2306-2315), followed by amino acidsISKRGGG (underlined below) and then by the mature Hu1D10 heavy chain.This plasmid did not include a selectable marker for Aspergillustransformation.

     MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSG(SEQ ID NO: 39)ADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASISKRGGGQVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWVRQSPGKGLEWIGVKWSGGSTEYNAAFISRLTISKDTSKNQVSLKLNSLTAADTAVYYCARNDRYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Plasmids pQ101 and pCL17 were co-transformed into Aspergillus niger varawamori strain dgr246ΔGAP:pyr2- by the methods described in Examples 9and 12. The best Hu1D10 producing transformant (3-Hu1D10-20e) wasidentified and found to produce approximately 0.2 g/l of IgG1κ.

Plasmids pQ100 and pCL16 were co-transformed into Aspergillus niger varawamori strain dgr246ΔGAP:pyr2- by the methods described in Examples 9and 12. The best Hu1D10 producing transformant (2-Hu1D10-16b) wasidentified and found to produce approximately 0.2 g/l of IgG1κ.

Antibody was purified from culture supernatant of these transformants bythe methods described in Example 12. The purified antibody preparationobtained from strain 3-Hu1D10 was designated An-Hu1D10 and that fromstrain 2-Hu1D10-16b was designated An-3G-Hu1D10.

Example 17 Antibody Affinity and Avidity

The human Burkitt's lymphoma-derived cell line Raji (ATCC, Manassas,Va.), which expresses the HLA-DR β chain allotype recognized by Hu1D10(Kostelny et al., 2001, Int J Cancer, 93:556), was maintained inRPMI-1640 (Gibco BRL, Grand Island, N.Y.) containing 10% fetal bovineserum (FBS; HyClone, Logan, Utah) in a 7.5% CO₂ incubator. The affinityof Hu1D10 binding to HLA-DR β chains was determined by measuring theamount of antibody that bound to Raji cells. Raji cells (5×10⁵cells/test) were incubated with varying amounts (serial 2-fold dilutionsstarting at 1 μg/test) of control Hu1D10 (derived from the NS0 mousemyeloma cell line), An-Hu1D10 or An-3G-Hu1D10 for 30 min on ice in 100μl of FACS Staining Buffer (FSB; PBS containing 1% bovine serum albuminand 0.2% sodium azide). After incubation, cells were washed three timesin FSB and incubated with fluorescein isothiocyanate (FITC)-conjugatedAffiniPure goat anti human IgG antibodies (Jackson ImmunoResearch, WestGrove, Pa.) for additional 30 min on ice. The cells were washed threetimes with FSB and analyzed by flow cytometry using a FACScan (BectonDickinson, San Jose, Calif.). Antibody concentration (ng/test) wasplotted versus mean channel fluorescence (FIG. 12). A competitionbinding experiment was also performed in which a mixture of FITC-labeledNS0-Hu1D10 (0.25 μg/test) and competitor antibody (serial 2-folddilutions of control NS0-derived Hu1D10, An-Hu1D10 or An-3G-Hu1D10starting at 6.25 μg/test) in FSB was added to Raji cells (5×10⁵cells/test) in a final volume of 100 μl per test in duplicate. Allsamples were incubated on ice for 30 min. The cells were washed threetimes with FSB and analyzed by flow cytometry. Competitor concentration(ng/test) was plotted versus mean channel fluorescence (FIG. 13). Nosignificant difference was observed in the binding to Raji cells amongthe NS0-derived and Aspergillus-derived Hu1D10 antibodies (FIGS. 12 and13), indicating that the production of Hu1D10 in Aspergillus niger hadno measurable effect on the structure of its antigen binding site.

In addition, the avidity of Hu1D10 was measured by monitoring the degreeof apoptosis in a population of Raji cells (as determined by stainingwith FITC-Annexin V and propidium iodide; Vermes, I. et al., 1995, J.Immunol. Methods 184:38-51). To measure the ability control NS0-derivedHu1D10, An-Hu1D10 or An-3G-Hu1D10 antibodies to induce apoptosis, Rajicells resuspended at 5×10⁵ cells/ml in RPMI-1640 containing 10% FBS wereincubated with 2 μg antibody at 37° C. for 5 hr or 24 hr. Cells werethen washed three times in 1× binding buffer provided in the ApoptosisDetection Kit (Pharmingen, San Diego, Calif.) and stained withFITC-conjugated annexin V and propidium iodide according to themanufacturer's protocol. Cell death was determined by 2-color flowcytometry. Percent apoptosis was defined as the sum of the percentage ofannexin V staining cells and the percentage of annexin V and propidiumiodide staining cells. Relative cell fluorescence was analyzed onFACScan (FIG. 14).

No significant difference in ability to induce apoptosis was observedbetween NS0-Hu1D10, An-Hu1D10 or An-3G-Hu1D10 in these experiments.

Example 18 Antibody-Dependent Cellular Cytotoxicity (ADCC)

The ability of NS0-Hu1D10, An-Hu1D10 or An-3G-Hu1D10 to kill Raji cellsby ADCC was measured (Kostelny et al, 2001). ADCC was analyzed with theLDH Detection Kit (Roche Molecular Biochemicals, Indianapolis, Ind.)using human PBMC as effector cells (E) and Raji cells as target cells(T). Human peripheral blood mononuclear cells (PBMC) were isolated fromhealthy donors using Ficoll-Paque PLUS lymphocyte isolation solution(Amersham Biosciences, Uppsala, Sweden). Target and effector cells werewashed in RPMI-1640 (Gibco BRL) supplemented with 1% BSA and added to96-well U-bottom plates (Becton Dickinson) at an E:T ratio of 40:1.Hu1D10 antibodies were added to the wells at desired concentrations.After a 4 hr incubation at 37° C., all plates were centrifuged andcell-free supernatants were incubated with LDH reaction mixture inseparate 96-well flat-bottom plates for 30 min at 25° C. The absorbanceof reaction samples was measured at 490 nm. Antibody-independentcellular cytotoxicity (AICC) was measured by adding effector and targetcells in the absence of antibodies. Spontaneous release (SR) wasmeasured by adding only target or effector cells. Maximal release (MR)was measured by adding 2% Triton-X100 to target cells. Percent lysis wasdetermined by the following equation: {(LDH release of sample−SR ofeffector cells−SR of target cells)/(MR of target cells−SR of targetcells)}×100. Each condition was examined in duplicate.

Human PBMC from two different donors were used in the analysis. Withdonor 1 (FIG. 15, left panel), the maximal cytotoxicity level reachednearly 40% with either of the three Hu1D10 antibodies. In thisparticular experiment, the Aspergillus-derived An-Hu1D10 inducedcytotoxicity slightly better than the other two Hu1D10 antibodies.Between the NS0-derived Hu1D10 and the Aspergillus-derived An-3G-Hu1D10antibodies, however, there was no significant difference in induction ofcytotoxicity. With donor 2 (FIG. 15, right panel), the maximalcytotoxicity levels were between 15 to 20% with the three Hu1D10antibodies. In this experiment, the An-Hu1D10 antibody was not as activein inducing cytotoxicity as the other two Hu1D10 antibodies, althoughthe difference among the three antibodies was minimal. These resultsclearly indicate that the Aspergillus-derived Hu1D10 antibodies exhibitADCC activities.

Example 19 Pharmacokinetics

An in vivo rat study was performed in order to compare thepharmacokinetics of trastuzumab purified from A. niger strain2-LC/HC-38b with that of trastuzumab (Herceptin) purchased fromGenentech Inc., South San Francisco.

Two groups of Sprague Dawley rats (weight range of approximately 250-300g) received a 2 mg/kg IV bolus dose of A. niger-derived trastuzumab(N=3) or of the commercial trastuzumab (N=4). Animals were dosedaccording to individual body weight using trastuzumab preparations thathad been diluted to a final concentration of 0.9 mg/mL. Blood for serum(0.5 mL/sample) was collected at 0, 1, 4, 8, 24, 48, 72 and 96 hours and7, 12, and 14 days post-dose. Serum was prepared by centrifugation ofthe blood sample within 30 minutes of collection. The serum was decantedand the serum samples were stored on ice until transfer for storage at−80° C. Human IgG1 levels in these serum samples were measured by ELISAas described above. The serum concentration versus time profiles of thefungal-derived and commercial trastuzumabs are shown in FIG. 16. Anoncompartmental analysis of the data was performed (Table 1). Theparameters from this analysis as well as the serum concentration versustime profiles of trastuzumab from 2 LC/HC-38b and the commercial sourcewere similar. Given the long survival time in the serum of bothcommercial trastuzumab and the antibody from 2 LC/HC-38b an accurateestimate of half-life could not be determined in this 14 day study.However, the parameters commonly used to evaluate bioequivalence, namelyC_(max) (the mean peak concentration of antibody in the serum) andAUC_(last) (area under the concentration-time curve), were comparablefor the 2 LC/HC 38b and mammalian cell-derived trastuzumabs. Theseresults indicated that the fungal expression of trastuzumab did notaffect the pharmacokinetic disposition of the antibody in vivo.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

TABLE 1 Pharmacokinetics of CHO-derived and Aspergillus- derivedtrastuzumab Meaned parameters from individual animals Fungal CHO Ab AbParameter Units n = 4 n = 3 Cmax ug/mL 49.5(6.2) 48.3(3)No_points_Lambda_z 8.25(1.5) 4.67(1.5) AUClast day * ug/mL  153(4.4) 143(11) HL_Lambda_z day 11.1(3.3)   15(2) AUCINF_obs day * ug/mL 297(45)  285(5.4) AUC_% Extrap_obs % 47.7(6.9) 49.8(3) Vz_obs mL/kg 106(16)  152(23)

1. A process for producing an immunoglobulin molecule or animmunologically functional immunoglobulin fragment comprising at leastthe variable domains of the immunoglobulin heavy and light chains, in ahost filamentous fungus, comprising the steps of: a. transforming saidhost with a first expression vector containing a fusion DNA constructencoding a first fusion polypeptide comprising, from a 5′ end of saidDNA construct, first, second, third and fourth nucleic acids, whereinsaid first nucleic acid encodes a signal polypeptide functional as asecretory sequence in the host, said second nucleic acid encodes asecreted polypeptide or functional portion thereof normally secretedfrom a filamentous fungus, said third nucleic acid encodes a cleavablelinker and said fourth nucleic acid encodes an immunoglobulin lightchain or fragment thereof; b. transforming said host with a secondexpression vector containing a fusion nucleic acid encoding a secondfusion polypeptide comprising, from a 5′ end of said fusion nucleicacid, first, second, third and fourth nucleic acids, wherein said firstnucleic acid encodes a signal polypeptide functional as a secretorysequence in the host, said second nucleic acid encodes a secretedpolypeptide or functional portion thereof normally secreted from afilamentous fungus, said third nucleic acid encodes a cleavable linkerand said fourth nucleic acid encodes an immunoglobulin heavy chain orfragment thereof; c. growing said host under conditions which permitexpression of said fusion DNA construct and said fusion nucleic acidsequences to cause the expression of the first fusion and the secondfusion polypeptides; and d. isolating said immunoglobulin molecule orimmunologically functional immunoglobulin fragment comprising at leastthe variable domains of the immunoglobulin heavy and lights chains;wherein the secreted polypeptide or functional portion thereof in (a)and (b) is glucoamylase, and the cleavable linker in (a) and (b) iscleavable by a native filamentous fungus protease, and wherein theimmunoglobulin or immunologically functional immunoglobulin fragment hasan amino acid substitution N297Q in the heavy chain, and theimmunoglobulin has reduced glycosylation due to the amino acidsubstitution N297Q in the heavy chain.
 2. The process of claim 1,wherein the immunoglobulin molecule or fragment thereof is secreted. 3.The process according to claim 1, wherein the host filamentous fungus isan Aspergillus, Neurospora, or Fusarium.
 4. The process according toclaim 3, wherein the host filamentous fungus is an Aspergillus.
 5. Theprocess according to claim 4, wherein the host filamentous fungus is anAspergillus niger.
 6. The process according to claim 1, wherein theimmunoglobulin has a fungal glycosylation pattern.
 7. The processaccording to claim 6, wherein the immunoglobulin has a high mannoseglycosylation pattern.
 8. The process according to claim 1, wherein thetransformation of step a) and step b) is a co-transformation.
 9. Theprocess according to claim 1, wherein the transformation of step a) andstep b) are sequential transformations.
 10. The host filamentous fungalcell transformed by the process of claim
 1. 11. The process according toclaim 1, wherein the cleavable linker in (a) and (b) is the KEX2 site.12. The process according to claim 1, wherein the host filamentousfungus is an Aspergillus, Neurospora, Fusarium, Trichoderma,Cephalosporium, Penicillium or Chrysosporium and wherein the secretedpolypeptide or functional portion thereof in (a) and (b) is glucoamylasefrom Aspergillus niger.